Methods and compositions for sequencing complementary polynucleotides

ABSTRACT

Disclosed herein, inter alia, are substrates, kits, and efficient methods of preparing and sequencing two or more regions of a double-stranded polynucleotide.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/147,167, filed Feb. 8, 2021; U.S. Provisional Application No.63/163,638, filed Mar. 19, 2021; and U.S. Provisional Application No.63/183,585, filed May 3, 2021; each of which are incorporated herein byreference in their entirety and for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The Sequence Listing titled 051385-542001US_SEQUENCE_LISTING_ST25.TXT,was created on Feb. 7, 2022 in machine format IBM-PC, MS-Windowsoperating system, is 17,892 bytes in size, and is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND

Genetic analysis is taking on increasing importance in modem society asa diagnostic, prognostic, and as a forensic tool. DNA sequencing is afundamental tool in biological and medical research; it is an essentialtechnology for the paradigm of personalized precision medicine. Sangersequencing, where the sequence of a nucleic acid is determined byselective incorporation and detection of dideoxynucleotides, enabled themapping of the first human reference genome. While this methodology isstill useful for validating newer sequencing technologies, efforts tosequence and assemble genomes using the Sanger method are an expensiveand laborious undertaking, requiring specialized equipment andexpertise. Next generation sequencing (NGS) methodologies make use ofsimultaneously sequencing millions of fragments of nucleic acids in asingle run. However, traditional next generation sequencing still hasshortcomings, such as challenges with detecting rare sequence variantsin the context of polymerase errors.

BRIEF SUMMARY

In view of the foregoing, innovative approaches to address issues withexisting sequencing technologies are needed. Disclosed herein aresolutions to these and other problems in the art which, in embodiments,increase the fidelity and accuracy of high throughput sequencingmethods. In certain embodiments, the compositions and methods providedherein reduce the amount of nucleic acid manipulation and duplicationrequired by traditional NGS techniques. Prior to the present disclosure,cluster-based sequencing processes would include cleaving and removingone strand from double-stranded molecules in a cluster before generatinga first read, without which the second strand would effectively competewith hybridization of the sequencing primer. Generating a sequencingread for the second (cleaved) strand would then require creating a newcomplementary strand from the sequenced first strand (i.e., a new secondstrand). In accordance with various embodiments, the methods disclosedherein permit obtaining sequence information (i.e., reading) from theoriginal first and second strands (e.g., original strands from theinitial cluster amplification, or amplicons), thereby reducing the time,reagents, expense, and risk of polymerase errors inherent in previousmethods.

In an aspect is a substrate (e.g., a solid support) including a firstpolynucleotide attached to the substrate; a second polynucleotideattached to the substrate, wherein the second polynucleotide includes acomplementary sequence to the first polynucleotide; and a thirdpolynucleotide hybridized to the second polynucleotide. In embodiments,the third polynucleotide is not covalently attached to the substrate.

In an aspect is provided a method of sequencing a templatepolynucleotide. In embodiments, the method includes: generating adouble-stranded amplification product including a first strandhybridized to a second strand, wherein (i) the double-strandedamplification product includes the template polynucleotide or complementthereof, and (ii) the first strand and second strand are both attachedto a solid support; generating a first invasion strand hybridized to thesecond strand by hybridizing one or more invasion primers to the secondstrand, and extending the one or more invasion primers; generating afirst sequencing read by hybridizing one or more sequencing primers tothe first strand, and extending the one or more first sequencingprimers. In embodiments, the invasion primer is not covalently attachedto the solid support.

In an aspect is provided a method of generating a template for nucleicacid sequencing reaction. In embodiments, the method includes providinga solid support including a plurality of immobilized oligonucleotideprimers attached to the solid support via a linker, wherein theplurality of oligonucleotide primers include a plurality of forwardprimers and a plurality of reverse primers, amplifying a templatenucleic acid by using the oligonucleotide primers attached to the solidsupport to generate a plurality of double-stranded amplificationproducts, each double-stranded amplification product including a firststrand hybridized to a second strand, wherein (i) each double-strandedamplification product includes the template polynucleotide or complementthereof, and (ii) the first strand and second strand are both attachedto the solid support; and (iii) generating a first invasion strandhybridized to the second strand by hybridizing one or more invasionprimers to the second strand, and extending the one or more invasionprimers; thereby generating a template nucleic acid for a nucleic acidsequencing reaction. In embodiments, the method further includeshybridizing one or more sequencing primers to the first strand. Inembodiments, the invasion primer is not covalently attached to the solidsupport.

In another aspect is provided a method including: amplifying a templatenucleic acid by using a plurality of oligonucleotide primers attached toa solid support to generate a plurality of double-stranded amplificationproducts, each double-stranded amplification product including a firststrand hybridized to a second strand, wherein the first strand andsecond strand are both attached to the solid support; and generating afirst invasion strand hybridized to the second strand by hybridizing oneor more invasion primers to the second strand, and extending the one ormore invasion primers to produce a single-stranded first strand. Inembodiments, the invasion primer is not covalently attached to the solidsupport.

In an aspect is provided a method of sequencing a double-strandedpolynucleotide including a first strand hybridized to a second strand,wherein the first strand and second strand are both attached to a solidsupport, the method including: i) hybridizing an invasion primer to thesecond strand and extending the invasion primer with a polymerase,thereby generating an invasion strand; ii) hybridizing a sequencingprimer to the first strand; iii) incorporating one or more nucleotidesinto the sequencing primer with a polymerase to create an extensionstrand; and iv) detecting the one or more incorporated nucleotides so asto identify each incorporated nucleotide in the extension strand,thereby sequencing the first strand of the double-strandedpolynucleotide.

In an aspect is provided a method of forming a plurality ofsingle-stranded polynucleotides attached to a solid support, the methodincluding: contacting a plurality of double-stranded polynucleotidesincluding a first strand hybridized to a second strand with a pluralityof invasion primers, wherein the first strand and the second strand areattached to the solid support; hybridizing one or more invasion primersto the second strand; and extending one or more invasion primershybridized to the second strand with a polymerase to generate one ormore invasion strands, displacing the first strand, thereby forming aplurality of single-stranded polynucleotides attached to the solidsupport.

In another aspect is provided a method of sequencing a templatepolynucleotide, the method including: generating a double-strandedamplification product including a first strand hybridized to a secondstrand, wherein (i) the double-stranded amplification product includesthe template polynucleotide or complement thereof, and (ii) the firststrand and second strand are both attached to a solid support;generating a first invasion strand hybridized to the second strand byhybridizing an invasion primer to the second strand, and extending theinvasion primer, wherein the invasion primer is not covalently attachedto the solid support; and generating a first sequencing read byhybridizing one or more sequencing primers to the first strand, andextending the one or more first sequencing primers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate an embodiment of paired-strand sequencing bystrand invasion of an invasion primer at the 3′ end of a first strand ofa duplex, followed by runoff extension of the invasion primer by astrand-displacing polymerase. The hashed boxes on each end represent apolymer scaffold that is anchored to a solid support, such as glass orsilicon support. FIG. 1A illustrates two dsDNA duplex strands, eachduplex having a first strand hybridized to a second strand, and eachstrand is attached to the solid support. By way of simplification, onlyone duplex is shown, however it is understood that a plurality ofduplexes (double-stranded amplification products) are present on thesolid support, typically in a plurality of localized monoclonalclusters. An invasion oligonucleotide (also referred to herein as aninvasion primer or invasion oligo) anneals at the 3′ end of one of thestrands. After extension of the invasion oligonucleotide has beencompleted, one strand of the initial dsDNA molecule is nowsingle-stranded and available for a first sequencing read, as shown inFIG. 1B. Also illustrated in FIG. 1B, the sequenced strand mayoptionally be cleaved at a cleavable site (represented as ‘X’) andremoved, thus leaving the complementary strand available for sequencing.For clarity in the figure, a single X is depicted, however it isunderstood that the cleavable site may include multiple chemical,enzymatic, or photochemical entities capable of being cleaved.

FIGS. 2A-2B show fluorescence images and data showing monoclonal DNAclusters examined under four conditions: 1) non-cleaved,invaded/extended; 2) invasion oligo, no extension; 3) non-cleaved, noinvasion/extension; 4) cleaved control. FIG. 2A shows the fluorescentimages for each of the four conditions, as well as a cartoonillustration of the duplex and fluorescently labeled probe under eachrespective image. FIG. 2B shows the median fluorescent signal and numberof identified features for each condition. Condition 4 is a positivecontrol whereby the clusters were converted into ssDNA by cleaving andremoving one of the strands from the flow cell surface, followed bylabeling the resulting ssDNA molecules with a complementary FAM-labeledDNA probe. Condition 3 is a negative control whereby the ampliconclusters are not subjected to an invasion primer nor extensionconditions. Condition 2 reveals that an invasion primer is capable ofinvading the double-stranded amplification product, but withoutextension of the invasion primer, the amplicons are not completelyaccessible to FAM-labeled probes. Condition 1 shows dsDNA clusters thatwere not cleaved, but were subjected to the methods described herein,such as strand invasion and extension, followed by hybridization of acomplementary FAM-labeled DNA probe to the liberated ssDNA strand. Theprobe is only able to hybridize if a complementary single-strandedregion is available. Following probe excitation and image acquisition,Condition 1 and condition 4 show the presence of punctate clustersindicative of successful ssDNA formation using the methods describedherein.

FIG. 3 reports the quality scores for sequencing the positive controlCondition 4 (i.e., intentionally formed ssDNA clusters as shown in FIGS.2A-2B) and for dsDNA clusters subjected to the methods described herein.

FIGS. 4A-4B illustrate an embodiment of strand invasion using aninvasion primer that contains peptide nucleic acids (PNAs) into dsDNAclusters. PNA oligos can invade into dsDNA at low ionic strength (<25 mMNaCl) conditions. As described herein, the PNA-containing invasionprimer is designed to invade at the common adapter sequence of the 5′end of one of the solid phase-bound amplicons. This, in turn, makes thedisplaced complementary 3′ DNA end on the complementary strandaccessible for binding with another invasion oligonucleotide, referredto as a runoff primer in FIG. 4B, that can be extended by astrand-displacing DNA polymerase. At the end of that process, one of thetwo strands of the initially dsDNA cluster is now single-stranded andaccessible for hybridization with a sequencing primer for a firstsequencing read. The sequenced strand may further be cleaved at acleavable site (represented as ‘X’) and removed, thus leaving thecomplementary strand available for sequencing.

FIGS. 5A-5B illustrate an embodiment of strand invasion into dsDNAmonoclonal clusters by using a recombinase and an invasionoligonucleotide. The pre-synaptic filament (alternatively referred to asa pre-synaptic complex), consisting of an invasion oligonucleotidecomplexed with recombinase enzymes searches dsDNA fragments forhomology. The invasion oligonucleotide can be inserted to itscomplementary sequence in the dsDNA amplicons, after which the invasionoligonucleotide can be extended by a strand-displacing polymerase. Thisrenders one of the two strands of the original dsDNA amplicon availablefor hybridization of a sequencing primer to initiate the SBS process.The sequenced strand may further be cleaved at a cleavable site(represented as ‘X’) and removed, thus leaving the complementary strandavailable for sequencing, as illustrated in FIG. 5B.

FIG. 6 shows a graph depicting the GC coverage bias dependence oninvasion-reaction conditions. Shown in the graph are results for a100×150 bp paired-strand sequencing run on dsDNA clusters that weresubjected to the methods described herein. The GC graph is for the first100 cycles of the 100×150 bp paired-strand sequencing experiment.Utilizing one invasion-reaction mixture (e.g., 30% DMSO only) skews thesequencing reads to cover 40-80% GC content. Alternating cycles of twoinvasion-reaction mixtures, that is a first invasion-reaction mixturecontaining 30% DMSO and a second invasion-reaction mixture containing 5%DMSO, results in greater coverage of all GC content.

FIGS. 7A-7B are graphs depicting the accuracy per cycle for a firstsequencing read (FIG. 7A) and a second sequencing read (FIG. 7B).

FIGS. 8A-8D illustrate an embodiment of paired-strand sequencing bystrand invasion of an invasion primer at the 3′ end of a first strand ofa duplex, followed by runoff extension of the invasion primer by astrand-displacing polymerase. FIG. 8A illustrates an invasion primerannealed to the 3′ end of one of the strands. In embodiments, theinvasion primer includes one or more phosphorothioate group(s) towardsthe 5′ end to protect the invasion primer from 5′ to 3′ exonucleasedigestion. In embodiments, the invasion primer also includes a cleavablesite (also referred to herein as a scissile linkage). For example, asdepicted as a ‘U’ in FIGS. 8A-8C, the cleavable site may be adeoxyuracil (dU) towards the 3′ end of the invasion oligo. After runoffextension of the invasion oligonucleotide has been completed, one strandof the initial dsDNA molecule is now single-stranded and available for afirst sequencing read, as shown in FIG. 8B. This renders one of the twostrands of the original dsDNA amplicon available for hybridization of asequencing primer to initiate the SBS process. The sequenced strand mayfurther optionally be cleaved at a cleavable site (represented as ‘X’)and removed, thus leaving the complementary strand available forsequencing, as illustrated in FIG. 8B. Subsequently, the 3′ end of theinvasion primer may be cleaved at a cleavable site (e.g., nicking the dUusing suitable conditions), leaving behind a 5′-phosphate in theinvasion strand that can subsequently be degraded with a 5′ to 3′exonuclease, allowing for the invasion primer to serve as a sequencingprimer for the second strand, as illustrated in FIGS. 8C-8D.

FIGS. 9A-9D illustrate an embodiment of paired-strand sequencing bystrand invasion of an invasion primer at the 3′ end of a first strand ofa duplex, followed by runoff extension of the invasion primer by astrand-displacing polymerase. FIG. 9A illustrates an invasion primerannealed to the 3′ end of one of the strands. In embodiments, theinvasion primer includes one or more phosphorothioate group(s) towardsthe 5′ end to protect the invasion primer from 5′ to 3′ exonucleasedigestion. In embodiments, the invasion primer also includes a cleavablesite (also referred to herein as a scissile linkage). For example, asdepicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towardsthe 3′ end of the invasion oligo. After runoff extension (i.e.,extension to a sufficient length) of the invasion oligonucleotide hasbeen completed, one strand of the initial dsDNA molecule is nowsingle-stranded and available for a first sequencing read, as shown inFIG. 9B. This renders one of the two strands of the original dsDNAamplicon available for hybridization of a sequencing primer to initiatethe SBS process. The sequenced strand may further be extended withnative dNTPs to complete the extension of the sequenced strand, asillustrated in FIG. 9B as the solid line beyond the star. Furtherextending the sequencing primer with unmodified nucleotides eliminatesany remaining single-stranded region. Subsequently, the 3′ end of theinvasion primer may be cleaved at a cleavable site (e.g., cleaving thedU using a uracil DNA glycosylase or formamidopyrimidine DNA glycosylase(Fpg) as described herein), leaving behind a 5′-phosphate in theextended part of the invasion primer that can subsequently be degradedwith a 5′ to 3′ exonuclease, allowing for the invasion primer to serveas a sequencing primer for the second strand, as illustrated in FIGS.9C-9D.

FIGS. 10A-10D illustrate an embodiment of paired-strand sequencing bystrand invasion of an invasion primer at the 3′ end of a first strand ofa duplex, followed by runoff extension of the invasion primer by astrand-displacing polymerase. FIG. 10A illustrates an invasion primerannealed to the 3′ end of one of the strands. In embodiments, theinvasion primer includes one or more phosphorothioate group(s) towardsthe 5′ end to protect the invasion primer from 5′ to 3′ exonucleasedigestion. In embodiments, the invasion primer also includes a cleavablesite (also referred to herein as a scissile linkage). For example, asdepicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towardsthe 3′ end of the invasion oligo. Following runoff extension of theinvasion oligonucleotide, one strand of the initial dsDNA molecule isnow single-stranded and available for a first sequencing read, as shownin FIG. 10B. This renders one of the two strands of the original dsDNAamplicon available for hybridization of a sequencing primer to initiatethe SBS process. The sequenced strand may further be extended with oneor more dideoxynucleotide triphosphates (ddNTPs) to prevent furtherextension, as illustrated in FIG. 10B as the hexagon. Further extendingthe sequencing primer with ddNTPs eliminates any prevents any furtherextension. Subsequently, the cleavable site at the 3′ end of theinvasion primer may be cleaved (e.g., the dU), leaving behind a5′-phosphate in the extended part of the invasion primer that cansubsequently be degraded with a 5′ to 3′ exonuclease, allowing for theinvasion primer to serve as a sequencing primer for the second strand,as illustrated in FIGS. 10C-10D.

FIGS. 11A-11D illustrate an embodiment of paired-strand sequencing bystrand invasion of an invasion primer at the 3′ end of a first strand ofa duplex, followed by runoff extension of the invasion primer by astrand-displacing polymerase. FIG. 11A illustrates an invasion primerannealed to the 3′ end of one of the strands. In embodiments, theinvasion primer includes one or more phosphorothioate group(s) towardsthe 5′ end to protect the invasion primer from 5′ to 3′ exonucleasedigestion. In embodiments, the invasion primer also includes a cleavablesite (also referred to herein as a scissile linkage). For example, asdepicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towardsthe 3′ end of the invasion oligo. Runoff extension of the invasionoligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP,leaving one strand of the initial dsDNA molecule single-stranded andavailable for a first sequencing read, as shown in FIG. 11B. Thesequenced strand may optionally further be cleaved at a cleavable site(represented as ‘X’) and removed, thus leaving the complementary strandavailable for sequencing, as illustrated in FIG. 11B. Subsequently, theinvasion strand may be nicked at internal scissile sites (e.g.,resulting from amplification with the dUTP), leaving behind small, lowTm fragments that may be denatured and removed under suitableconditions, as shown in FIG. 11C. Additionally, this cleavage anddenaturation step exposes the 3′ end of the invasion oligo, allowing forthe invasion primer to serve as a sequencing primer for the secondstrand, as illustrated in FIGS. 11C-11D.

FIGS. 12A-12E illustrate an embodiment of paired-strand sequencing bystrand invasion of an invasion primer at the 3′ end of a first strand ofa duplex, followed by runoff extension of the invasion primer by astrand-displacing polymerase. FIG. 12A illustrates an invasion primerannealed to the 3′ end of one of the strands. In embodiments, theinvasion primer includes one or more phosphorothioate group(s) towardsthe 5′ end to protect the invasion primer from 5′ to 3′ exonucleasedigestion. In embodiments, the invasion primer also includes a cleavablesite (also referred to herein as a scissile linkage). For example, asdepicted as a ‘U’, the cleavable site may be a deoxyuracil (dU) towardsthe 3′ end of the invasion oligo. Runoff extension of the invasionoligonucleotide is then performed with dUTP, dATP, dGTP, and dCTP,leaving one strand of the initial dsDNA molecule single-stranded andavailable for a first sequencing read, as shown in FIG. 12B. Once thefirst sequencing read has been obtained, the 3′ end of the firstsequencing read is capped by ddNTP incorporation. A second sequencingread is then obtained by annealing and extending a second sequencingprimer 3′ of the terminated first sequencing read. Subsequently, a ddNTPis incorporated into the 3′ end of the second sequencing read, andthereafter the invasion strand may be nicked at internal scissile sites(e.g., resulting from amplification with the dUTP), leaving behind smallfragments with exposed 5′ ends that may be removed under suitableconditions, for example, by lambda exonuclease digestion, as shown inFIGS. 12C-12D. This cleavage and removal step exposes the 3′ end of thesecond strand, making it available for a third sequencing read, as shownin FIG. 12E. Once the third sequencing read has been obtained, the 3′end of the third sequencing read is capped by ddNTP incorporation. Afourth sequencing read is then obtained by annealing and extending afourth sequencing primer 3′ of the terminated third sequencing read, asillustrated in FIG. 12E.

DETAILED DESCRIPTION

The aspects and embodiments described herein relate to sequencing apolynucleotide. In embodiments, as described herein, the methods relateto sequencing a first strand of a double-stranded polynucleotide, andoptionally sequencing the complement of first strand (i.e., the secondstrand) of the same double-stranded polynucleotide. The terms “cluster”and “colony” are used interchangeably throughout this application andrefer to a discrete site on a solid support comprised of a plurality ofimmobilized nucleic acid strands. The term “clustered array” refers toan array formed from such clusters or colonies.

I. Definitions

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference in their entireties. The practice of the technologydescribed herein will employ, unless indicated specifically to thecontrary, conventional methods of chemistry, biochemistry, organicchemistry, molecular biology, bioinformatics, microbiology, recombinantDNA techniques, genetics, immunology, and cell biology that are withinthe skill of the art, many of which are described below for the purposeof illustration. Examples of such techniques are available in theliterature. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY ANDMOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); andSambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition(2012). Methods, devices and materials similar or equivalent to thosedescribed herein can be used in the practice of this invention.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the disclosure, some preferred methods and materials aredescribed. Accordingly, the terms defined immediately below are morefully described by reference to the specification as a whole. It is tobe understood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context in which they are used by those of skill inthe art. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the singular terms “a”, “an”, and “the” include theplural reference unless the context clearly indicates otherwise.Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present.

As used herein, the term “control” or “control experiment” is used inaccordance with its plain and ordinary meaning and refers to anexperiment in which the subjects or reagents of the experiment aretreated as in a parallel experiment except for omission of a procedure,reagent, or variable of the experiment. In some instances, the controlis used as a standard of comparison in evaluating experimental effects.

As used herein, the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation, where for example digital information regarding two or morespecies is stored and can be used to determine that one or more of thespecies were co-located at a point in time. An association can also be aphysical association.

As used herein, the term “complementary” or “substantiallycomplementary” refers to the hybridization, base pairing, or theformation of a duplex between nucleotides or nucleic acids. For example,complementarity exists between the two strands of a double-stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA)or a sequence of nucleotides is capable of base pairing with arespective cognate nucleotide or cognate sequence of nucleotides. Whenreferring to a double-stranded polynucleotide including a first strandhybridized to a second strand, it is to be understood that each of theterms “first strand” and “second strand” refer to single-strandedpolynucleotides. As described herein and commonly known in the art thecomplementary (matching) nucleotide of adenosine (A) is thymidine (T)and the complementary (matching) nucleotide of guanosine (G) is cytosine(C). Thus, a complement may include a sequence of nucleotides that basepair with corresponding complementary nucleotides of a second nucleicacid sequence. The nucleotides of a complement may partially orcompletely match the nucleotides of the second nucleic acid sequence.Where the nucleotides of the complement completely match each nucleotideof the second nucleic acid sequence, the complement forms base pairswith each nucleotide of the second nucleic acid sequence. Where thenucleotides of the complement partially match the nucleotides of thesecond nucleic acid sequence only some of the nucleotides of thecomplement form base pairs with nucleotides of the second nucleic acidsequence. Examples of complementary sequences include coding andnon-coding sequences, wherein the non-coding sequence containscomplementary nucleotides to the coding sequence and thus forms thecomplement of the coding sequence. A further example of complementarysequences are sense and antisense sequences, wherein the sense sequencecontains complementary nucleotides to the antisense sequence and thusforms the complement of the antisense sequence. “Duplex” means at leasttwo oligonucleotides and/or polynucleotides that are fully or partiallycomplementary undergo Watson-Crick type base pairing among all or mostof their nucleotides so that a stable complex is formed. Complementarysingle stranded nucleic acids and/or substantially complementary singlestranded nucleic acids can hybridize to each other under hybridizationconditions, thereby forming a nucleic acid that is partially or fullydouble stranded. When referring to a double-stranded polynucleotideincluding a first strand hybridized to a second strand, it is understoodthat each of the first strand and the second strand are independentlysingle-stranded polynucleotides. All or a portion of a nucleic acidsequence may be substantially complementary to another nucleic acidsequence, in some embodiments. As referred to herein, “substantiallycomplementary” refers to nucleotide sequences that can hybridize witheach other under suitable hybridization conditions. Hybridizationconditions can be altered to tolerate varying amounts of sequencemismatch within complementary nucleic acids that are substantiallycomplementary. Substantially complementary portions of nucleic acidsthat can hybridize to each other can be 75% or more, 76% or more, 77% ormore, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more,83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% ormore, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more,94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99%or more complementary to each other. In some embodiments substantiallycomplementary portions of nucleic acids that can hybridize to each otherare 100% complementary. Nucleic acids, or portions thereof, that areconfigured to hybridize to each other often comprise nucleic acidsequences that are substantially complementary to each other.

As described herein, the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other, may have aspecified percentage of nucleotides that complement one another (e.g.,about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion). In embodiments, two sequences are complementary when they arecompletely complementary, having 100% complementarity. In embodiments,sequences in a pair of complementary sequences form portions of a singlepolynucleotide with non-base-pairing nucleotides (e.g., as in a hairpinor loop structure, with or without an overhang) or portions of separatepolynucleotides. In embodiments, one or both sequences in a pair ofcomplementary sequences form portions of longer polynucleotides, whichmay or may not include additional regions of complementarity.

As used herein, the term “contacting” is used in accordance with itsplain ordinary meaning and refers to the process of allowing at leasttwo distinct species (e.g. chemical compounds including biomolecules orcells) to become sufficiently proximal to react, interact or physicallytouch. However, the resulting reaction product can be produced directlyfrom a reaction between the added reagents or from an intermediate fromone or more of the added reagents that can be produced in the reactionmixture. The term “contacting” may include allowing two species toreact, interact, or physically touch, wherein the two species may be acompound, nucleic acid, a protein, or enzyme (e.g., a DNA polymerase).

As may be used herein, the terms “nucleic acid,” “nucleic acidmolecule,” “nucleic acid sequence,” “nucleic acid fragment” and“polynucleotide” are used interchangeably and are intended to include,but are not limited to, a polymeric form of nucleotides covalentlylinked together that may have various lengths, eitherdeoxyribonucleotides or ribonucleotides, or analogs, derivatives ormodifications thereof. Different polynucleotides may have differentthree-dimensional structures, and may perform various functions, knownor unknown. Non-limiting examples of polynucleotides include a gene, agene fragment, an exon, an intron, intergenic DNA (including, withoutlimitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA,ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, abranched polynucleotide, a plasmid, a vector, isolated DNA of asequence, isolated RNA of a sequence, a nucleic acid probe, and aprimer. Polynucleotides useful in the methods of the disclosure maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences. As may beused herein, the terms “nucleic acid oligomer” and “oligonucleotide” areused interchangeably and are intended to include, but are not limitedto, nucleic acids having a length of 200 nucleotides or less. In someembodiments, an oligonucleotide is a nucleic acid having a length of 2to 200 nucleotides, 2 to 150 nucleotides, 5 to 150 nucleotides or 5 to100 nucleotides. The terms “polynucleotide,” “oligonucleotide,” “oligo”or the like refer, in the usual and customary sense, to a linearsequence of nucleotides. Oligonucleotides are typically from about 5, 6,7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides in length, up toabout 100 nucleotides in length. In some embodiments, an oligonucleotideis a primer configured for extension by a polymerase when the primer isannealed completely or partially to a complementary nucleic acidtemplate. A primer is often a single stranded nucleic acid. In certainembodiments, a primer, or portion thereof, is substantiallycomplementary to a portion of an adapter. In some embodiments, a primerhas a length of 200 nucleotides or less. In certain embodiments, aprimer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides. In someembodiments, an oligonucleotide may be immobilized to a solid support.

As used herein, the terms “polynucleotide primer” and “primer” refers toany polynucleotide molecule that may hybridize to a polynucleotidetemplate, be bound by a polymerase, and be extended in atemplate-directed process for nucleic acid synthesis. The primer may bea separate polynucleotide from the polynucleotide template, or both maybe portions of the same polynucleotide (e.g., as in a hairpin structurehaving a 3′ end that is extended along another portion of thepolynucleotide to extend a double-stranded portion of the hairpin).Primers (e.g., forward or reverse primers) may be attached to a solidsupport (e.g., a polymer coated solid support). In embodiments, forwardprimers anneal to the antisense strand of the double-stranded DNA, whichruns from the 3′ to 5′ direction. Forward primers, for example, initiatethe synthesis of a gene in the 5′ to 3′ direction. In embodiments,reverse primers anneal to the sense strand of the double-stranded DNA,which runs from the 5′ to 3′ direction. Reverse primers, for example,initiate the synthesis of a gene in the 3′ to 5′ direction. A primer canbe of any length depending on the particular technique it will be usedfor. For example, PCR primers are generally between 10 and 40nucleotides in length. The length and complexity of the nucleic acidfixed onto the nucleic acid template may vary. In some embodiments, aprimer has a length of 200 nucleotides or less. In certain embodiments,a primer has a length of 10 to 150 nucleotides, 15 to 150 nucleotides, 5to 100 nucleotides, 5 to 50 nucleotides or 10 to 50 nucleotides. One ofskill can adjust these factors to provide optimum hybridization andsignal production for a given hybridization procedure. The primerpermits the addition of a nucleotide residue thereto, or oligonucleotideor polynucleotide synthesis therefrom, under suitable conditions. In anembodiment the primer is a DNA primer, i.e., a primer consisting of, orlargely consisting of, deoxyribonucleotide residues. The primers aredesigned to have a sequence that is the complement of a region oftemplate/target DNA to which the primer hybridizes. The addition of anucleotide residue to the 3′ end of a primer by formation of aphosphodiester bond results in a DNA extension product. The addition ofa nucleotide residue to the 3′ end of the DNA extension product byformation of a phosphodiester bond results in a further DNA extensionproduct. In another embodiment the primer is an RNA primer. Inembodiments, a primer is hybridized to a target polynucleotide. A“primer” is complementary to a polynucleotide template, and complexes byhydrogen bonding or hybridization with the template to give aprimer/template complex for initiation of synthesis by a polymerase,which is extended by the addition of covalently bonded bases linked atits 3′ end complementary to the template in the process of DNAsynthesis.

As used herein, the terms “invasion primer”, “invasion oligonucleotide”and “third polynucleotide” refer to a polynucleotide molecule that mayhybridize to a single-stranded nucleic acid sequence of adouble-stranded polynucleotide and be extended in a template-directedprocess (e.g., extended with a polymerase) for nucleic acid synthesis.In embodiments, an invasion primer hybridizes at or near the end of thesingle-stranded nucleic acid sequence (e.g., the 5′ end or the 3′ end),or the invasion primer hybridizes at an internal sequence. Extension ofan invasion primer results in the formation of an “invasion strand”complementary to either the first strand or the second strand of thedouble-stranded polynucleotide. This renders one of the two strands ofthe original dsDNA amplicon available for hybridization of a sequencingprimer to initiate the sequencing process. In embodiments, the invasionprimer includes locked nucleic acids (LNAs), Bis-locked nucleic acids(bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleicacids (BNAs), 2′-O-methyl RNA:DNA chimeric nucleic acids, minor groovebinder (MGB) nucleic acids, morpholino nucleic acids, C5-modifiedpyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioatenucleic acids, or combinations thereof. In embodiments, the invasionprimer includes phosphorothioate nucleic acids. In embodiments, theinvasion primer includes one or more locked nucleic acids (LNAs),2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalizedoligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs),peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC)nucleic acids. In embodiments, the invasion primer includes 10 to 15locked nucleic acids (LNAs). In embodiments, the invasion primerincludes a sequence described herein, for example within Table 1. Inembodiments, the invasion primer includes one or more phosphorothioatesat the 5′ end. In embodiments, the invasion primer includes one or moreLNAs at the 5′ end. In embodiments, the invasion primer includes two ormore consecutive LNAs at the 3′ end. In embodiments, the invasion primerincludes two or more consecutive LNAs at the 5′ end. In embodiments, theinvasion primer includes a plurality (e.g., 2 to 10) of syntheticnucleotides (e.g., LNAs) and a plurality (e.g., 2 to 10) canonical ornative nucleotides (e.g., dNTPs). In embodiments, the invasion primerincludes one or more (e.g., 2 to 5) deoxyuracil nucleobases (dU). Inembodiments, the one or more dU nucleobases are at or near the 3′ end ofthe invasion primer (e.g., within 5 nucleotides of the 3′ end). Inembodiments, the one or more dU nucleobases are distributed through theinvasion primer. In embodiments, the invasion primer includes from 5′ to3′ a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids,followed by a plurality of synthetic nucleotides (e.g., LNAs), and aplurality (e.g., 2 to 10) of canonical bases. In some embodiments, theinvasion primer includes a plurality of canonical bases, wherein thecanonical bases terminate (i.e., at the 3′ end) with a deoxyuracilnucleobase (dU). In embodiments, the invasion primer is about 10 to 100nucleotides in length. In embodiments, the invasion primer is about 15to about 40 nucleotides in length. In embodiments, the calculated orpredicted melting temperature (Tm) of the invasion primer is about 70°C. to about 95° C. In embodiments, the calculated or predicted meltingtemperature (Tm) of the invasion primer is about 75° C. to about 85° C.In embodiments, the calculated or predicted melting temperature (Tm) ofthe invasion primer is 75° C. to 85° C.

As used herein, the terms “solid support” and “substrate” and “solidsurface” are used interchangeably and refers to discrete solid orsemi-solid surfaces to which a plurality of nucleic acid (e.g., primers)may be attached. A solid support may encompass any type of solid,porous, or hollow sphere, ball, cylinder, or other similar configurationcomposed of plastic, ceramic, metal, or polymeric material (e.g.,hydrogel) onto which a nucleic acid may be immobilized (e.g., covalentlyor non-covalently). A solid support may comprise a discrete particlethat may be spherical (e.g., microspheres) or have a non-spherical orirregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical,oblong, or disc-shaped, and the like. Solid supports may be in the formof discrete particles, which alone does not imply or require anyparticular shape. The term “particle” means a small body made of a rigidor semi-rigid material. The body can have a shape characterized, forexample, as a sphere, oval, microsphere, or other recognized particleshape whether having regular or irregular dimensions. As used herein,the term “discrete particles” refers to physically distinct particleshaving discernible boundaries. The term “particle” does not indicate anyparticular shape. The shapes and sizes of a collection of particles maybe different or about the same (e.g., within a desired range ofdimensions, or having a desired average or minimum dimension). Aparticle may be substantially spherical (e.g., microspheres) or have anon-spherical or irregular shape, such as cubic, cuboid, pyramidal,cylindrical, conical, oblong, or disc-shaped, and the like. Inembodiments, the particle has the shape of a sphere, cylinder,spherocylinder, or ellipsoid. Discrete particles collected in acontainer and contacting one another will define a bulk volumecontaining the particles, and will typically leave some internalfraction of that bulk volume unoccupied by the particles, even whenpacked closely together. In embodiments, cores and/or core-shellparticles are approximately spherical. As used herein the term“spherical” refers to structures which appear substantially or generallyof spherical shape to the human eye, and does not require a sphere to amathematical standard. In other words, “spherical” cores or particlesare generally spheroidal in the sense of resembling or approximating toa sphere. In embodiments, the diameter of a spherical core or particleis substantially uniform, e.g., about the same at any point, but maycontain imperfections, such as deviations of up to 1, 2, 3, 4, 5 or upto 10%. Because cores or particles may deviate from a perfect sphere,the term “diameter” refers to the longest dimension of a given core orparticle. Likewise, polymer shells are not necessarily of perfectuniform thickness all around a given core. Thus, the term “thickness” inrelation to a polymer structure (e.g., a shell polymer of a core-shellparticle) refers to the average thickness of the polymer layer.

A solid support may further comprise a polymer or hydrogel on thesurface to which the primers are attached (e.g., the primers arecovalently attached to the polymer, wherein the polymer is in directcontact with the solid support). Exemplary solid supports include, butare not limited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefin copolymers, polyimides etc.), nylon, ceramics,resins, Zeonor, silica or silica-based materials including silicon andmodified silicon, carbon, metals, inorganic glasses, optical fiberbundles, photopatternable dry film resists, UV-cured adhesives andpolymers. The solid supports for some embodiments have at least onesurface located within a flow cell. The solid support, or regionsthereof, can be substantially flat. The solid support can have surfacefeatures such as wells, pits, channels, ridges, raised regions, pegs,posts or the like. The term solid support is encompassing of a substrate(e.g., a flow cell) having a surface comprising a polymer coatingcovalently attached thereto. In embodiments, the solid support is a flowcell. The term “flow cell” as used herein refers to a chamber includinga solid surface across which one or more fluid reagents can be flowed.Examples of flow cells and related fluidic systems and detectionplatforms that can be readily used in the methods of the presentdisclosure are described, for example, in Bentley et al., Nature456:53-59 (2008). In certain embodiments a substrate comprises a surface(e.g., a surface of a flow cell, a surface of a tube, a surface of achip, surface of a particle), for example a metal surface (e.g., steel,gold, silver, aluminum, silicon and copper). In some embodiments asubstrate (e.g., a substrate surface) is coated and/or comprisesfunctional groups and/or inert materials. In certain embodiments asubstrate comprises a bead, a chip, a capillary, a plate, a membrane, awafer (e.g., silicon wafers), a comb, or a pin for example. In someembodiments a substrate comprises a bead and/or a nanoparticle. Asubstrate can be made of a suitable material, non-limiting examples ofwhich include a plastic or a suitable polymer (e.g., polycarbonate,poly(vinyl alcohol), poly(divinylbenzene), polystyrene, polyamide,polyester, polyvinylidene difluoride (PVDF), polyethylene, polyurethane,polypropylene, and the like), borosilicate, silica, nylon, Wang resin,Merrifield resin, metal (e.g., iron, a metal alloy, sepharose, agarose,polyacrylamide, dextran, cellulose and the like or combinations thereof.In some embodiments a substrate comprises a magnetic material (e.g.,iron, nickel, cobalt, platinum, aluminum, and the like). In certainembodiments a substrate comprises a magnetic bead (e.g., DYNABEADS®,hematite, AMPure XP). Magnets can be used to purify and/or capturenucleic acids bound to certain substrates (e.g., substrates comprising ametal or magnetic material).

As used herein, the term “polymer” refers to macromolecules having oneor more structurally unique repeating units. The repeating units arereferred to as “monomers,” which are polymerized for the polymer.Typically, a polymer is formed by monomers linked in a chain-likestructure. A polymer formed entirely from a single type of monomer isreferred to as a “homopolymer.” A polymer formed from two or more uniquerepeating structural units may be referred to as a “copolymer.” Apolymer may be linear or branched, and may be random, block, polymerbrush, hyperbranched polymer, bottlebrush polymer, dendritic polymer, orpolymer micelles. The term “polymer” includes homopolymers, copolymers,tripolymers, tetra polymers and other polymeric molecules made frommonomeric subunits. Copolymers include alternating copolymers, periodiccopolymers, statistical copolymers, random copolymers, block copolymers,linear copolymers and branched copolymers. The term “polymerizablemonomer” is used in accordance with its meaning in the art of polymerchemistry and refers to a compound that may covalently bind chemicallyto other monomer molecules (such as other polymerizable monomers thatare the same or different) to form a polymer. Polymers can behydrophilic, hydrophobic, or amphiphilic, as known in the art. Thus,“hydrophilic polymers” are substantially miscible with water andinclude, but are not limited to, polyethylene glycol and the like.“Hydrophobic polymers” are substantially immiscible with water andinclude, but are not limited to, polyethylene, polypropylene,polybutadiene, polystyrene, polymers disclosed herein, and the like.“Amphiphilic polymers” have both hydrophilic and hydrophobic propertiesand are typically copolymers having hydrophilic segment(s) andhydrophobic segment(s). Polymers include homopolymers, randomcopolymers, and block copolymers, as known in the art. The term“homopolymer” refers, in the usual and customary sense, to a polymerhaving a single monomeric unit. The term “copolymer” refers to a polymerderived from two or more monomeric species. The term “random copolymer”refers to a polymer derived from two or more monomeric species with nopreferred ordering of the monomeric species. The term “block copolymer”refers to polymers having two or homopolymer subunits linked by covalentbond. Thus, the term “hydrophobic homopolymer” refers to a homopolymerwhich is hydrophobic. The term “hydrophobic block copolymer” refers totwo or more homopolymer subunits linked by covalent bonds and which ishydrophobic.

As used herein, the term “hydrogel” refers to a three-dimensionalpolymeric structure that is substantially insoluble in water, but whichis capable of absorbing and retaining large quantities of water to forma substantially stable, often soft and pliable, structure. Inembodiments, water can penetrate in between polymer chains of a polymernetwork, subsequently causing swelling and the formation of a hydrogel.In embodiments, hydrogels are super-absorbent (e.g., containing morethan about 90% water) and can be comprised of natural or syntheticpolymers.

The term “surface” is intended to mean an external part or externallayer of a substrate. The surface can be in contact with anothermaterial such as a gas, liquid, gel, polymer, organic polymer, secondsurface of a similar or different material, metal, or coating. Thesurface, or regions thereof, can be substantially flat. The substrateand/or the surface can have surface features such as wells, pits,channels, ridges, raised regions, pegs, posts or the like.

As used herein, the terms “cluster” and “colony” are usedinterchangeably to refer to a discrete site on a solid support thatincludes a plurality of immobilized polynucleotides and a plurality ofimmobilized complementary polynucleotides. The term “clustered array”refers to an array formed from such clusters or colonies. In thiscontext the term “array” is not to be understood as requiring an orderedarrangement of clusters. The term “array” is used in accordance with itsordinary meaning in the art, and refers to a population of differentmolecules that are attached to one or more solid-phase substrates suchthat the different molecules can be differentiated from each otheraccording to their relative location. An array can include differentmolecules that are each located at different addressable features on asolid-phase substrate. The molecules of the array can be nucleic acidprimers, nucleic acid probes, nucleic acid templates or nucleic acidenzymes such as polymerases or ligases. Arrays useful in the inventioncan have densities that ranges from about 2 different features to manymillions, billions or higher. The density of an array can be from 2 toas many as a billion or more different features per square cm. Forexample an array can have at least about 100 features/cm², at leastabout 1,000 features/cm², at least about 10,000 features/cm², at leastabout 100,000 features/cm², at least about 10,000,000 features/cm², atleast about 100,000,000 features/cm², at least about 1,000,000,000features/cm², at least about 2,000,000,000 features/cm² or higher. Inembodiments, the arrays have features at any of a variety of densitiesincluding, for example, at least about 10 features/cm², 100features/cm², 500 features/cm², 1,000 features/cm², 5,000 features/cm²,10,000 features/cm², 50,000 features/cm², 100,000 features/cm²,1,000,000 features/cm², 5,000,000 features/cm², or higher.

Nucleic acids, including e.g., nucleic acids with a phosphorothioatebackbone, can include one or more reactive moieties. As used herein, theterm reactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

As used herein, the term “template polynucleotide” or “template nucleicacid” refers to any polynucleotide molecule that may be bound by apolymerase and utilized as a template for nucleic acid synthesis. Atemplate polynucleotide may be a target polynucleotide. In general, theterm “target polynucleotide” refers to a nucleic acid molecule orpolynucleotide in a starting population of nucleic acid molecules havinga target sequence whose presence, amount, and/or nucleotide sequence, orchanges in one or more of these, are desired to be determined. Ingeneral, the term “target sequence” refers to a nucleic acid sequence ona single strand of nucleic acid. The terms “single strand” and “ssDNA”are used in accordance with its plain and ordinary meaning and refer toa single-stranded polynucleotide. The target sequence may be a portionof a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA,miRNA, rRNA, or others. The target sequence may be a target sequencefrom a sample or a secondary target such as a product of anamplification reaction. A target polynucleotide is not necessarily anysingle molecule or sequence. For example, a target polynucleotide may beany one of a plurality of target polynucleotides in a reaction, or allpolynucleotides in a given reaction, depending on the reactionconditions. For example, in a nucleic acid amplification reaction withrandom primers, all polynucleotides in a reaction may be amplified. As afurther example, a collection of targets may be simultaneously assayedusing polynucleotide primers directed to a plurality of targets in asingle reaction. As yet another example, all or a subset ofpolynucleotides in a sample may be modified by the addition of aprimer-binding sequence (such as by the ligation of adapters containingthe primer binding sequence), rendering each modified polynucleotide atarget polynucleotide in a reaction with the corresponding primerpolynucleotide(s). In the context of selective sequencing, “targetpolynucleotide(s)” refers to the subset of polynucleotide(s) to besequenced from within a starting population of polynucleotides.

In embodiments, a target polynucleotide is a cell-free polynucleotide.In general, the terms “cell-free,” “circulating,” and “extracellular” asapplied to polynucleotides (e.g. “cell-free DNA” (cfDNA) and “cell-freeRNA” (cfRNA)) are used interchangeably to refer to polynucleotidespresent in a sample from a subject or portion thereof that can beisolated or otherwise manipulated without applying a lysis step to thesample as originally collected (e.g., as in extraction from cells orviruses). Cell-free polynucleotides are thus unencapsulated or “free”from the cells or viruses from which they originate, even before asample of the subject is collected. Cell-free polynucleotides may beproduced as a byproduct of cell death (e.g. apoptosis or necrosis) orcell shedding, releasing polynucleotides into surrounding body fluids orinto circulation. Accordingly, cell-free polynucleotides may be isolatedfrom a non-cellular fraction of blood (e.g. serum or plasma), from otherbodily fluids (e.g. urine), or from non-cellular fractions of othertypes of samples.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

As used herein, the terms “analogue” and “analog”, in reference to achemical compound, refers to compound having a structure similar to thatof another one, but differing from it in respect of one or moredifferent atoms, functional groups, or substructures that are replacedwith one or more other atoms, functional groups, or substructures. Inthe context of a nucleotide, a nucleotide analog refers to a compoundthat, like the nucleotide of which it is an analog, can be incorporatedinto a nucleic acid molecule (e.g., an extension product) by a suitablepolymerase, for example, a DNA polymerase in the context of a nucleotideanalogue. The terms also encompass nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, which aresynthetic, naturally occurring, or non-naturally occurring, which havesimilar binding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, include, without limitation, phosphodiesterderivatives including, e.g., phosphoramidate, phosphorodiamidate,phosphorothioate (also known as phosphorothioate having double bondedsulfur replacing oxygen in the phosphate), phosphorodithioate,phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acid,phosphonoformic acid, methyl phosphonate, boron phosphonate, orO-methylphosphoroamidite linkages (see, e.g., see Eckstein,OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, Oxford UniversityPress) as well as modifications to the nucleotide bases such as in5-methyl cytidine or pseudouridine; and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, modified sugars, and non-ribosebackbones (e.g. phosphorodiamidate morpholino oligos or locked nucleicacids (LNA)), including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CARBOHYDRATEMODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. In embodiments, the internucleotide linkages in DNAare phosphodiester, phosphodiester derivatives, or a combination ofboth.

As used herein, a “native” nucleotide is used in accordance with itsplain and ordinary meaning and refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as may characterize anucleotide analog (e.g., a reversible terminating moiety). Examples ofnative nucleotides useful for carrying out procedures described hereininclude: dATP (2′-deoxyadenosine-5′-triphosphate); dGTP(2′-deoxyguanosine-5′-triphosphate); dCTP(2′-deoxycytidine-5′-triphosphate); dTTP(2′-deoxythymidine-5′-triphosphate); and dUTP(2′-deoxyuridine-5′-triphosphate). A “canonical” nucleotide is anunmodified nucleotide.

As used herein, the term “modified nucleotide” refers to nucleotidemodified in some manner. Typically, a nucleotide contains a single5-carbon sugar moiety, a single nitrogenous base moiety and 1 to threephosphate moieties. In embodiments, a nucleotide can include a blockingmoiety (alternatively referred to herein as a reversible terminatormoiety) and/or a label moiety. A blocking moiety on a nucleotideprevents formation of a covalent bond between the 3′ hydroxyl moiety ofthe nucleotide and the 5′ phosphate of another nucleotide. A blockingmoiety on a nucleotide can be reversible, whereby the blocking moietycan be removed or modified to allow the 3′ hydroxyl to form a covalentbond with the 5′ phosphate of another nucleotide. A blocking moiety canbe effectively irreversible under particular conditions used in a methodset forth herein. In embodiments, the blocking moiety is attached to the3′ oxygen of the nucleotide and is independently —NH₂, —CN, —CH₃, C₂-C₆allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃), or —CH₂N₃.In embodiments, the blocking moiety is attached to the 3′ oxygen of thenucleotide and is independently

A label moiety of a nucleotide can be any moiety that allows thenucleotide to be detected, for example, using a spectroscopic method.Exemplary label moieties are fluorescent labels, mass labels,chemiluminescent labels, electrochemical labels, detectable labels andthe like. One or more of the above moieties can be absent from anucleotide used in the methods and compositions set forth herein. Forexample, a nucleotide can lack a label moiety or a blocking moiety orboth. Examples of nucleotide analogues include, without limitation,7-deaza-adenine, 7-deaza-guanine, the analogues of deoxynucleotidesshown herein, analogues in which a label is attached through a cleavablelinker to the 5-position of cytosine or thymine or to the 7-position ofdeaza-adenine or deaza-guanine, and analogues in which a small chemicalmoiety is used to cap the OH group at the 3′-position of deoxyribose.Nucleotide analogues and DNA polymerase-based DNA sequencing are alsodescribed in U.S. Pat. No. 6,664,079, which is incorporated herein byreference in its entirety for all purposes.

The term “cleavable linker” or “cleavable moiety” as used herein refersto a divalent or monovalent, respectively, moiety which is capable ofbeing separated (e.g., detached, split, disconnected, hydrolyzed, astable bond within the moiety is broken) into distinct entities. Acleavable linker is cleavable (e.g., specifically cleavable) in responseto external stimuli (e.g., enzymes, nucleophilic/basic reagents,reducing agents, photo-irradiation, electrophilic/acidic reagents,organometallic and metal reagents, or oxidizing reagents). A chemicallycleavable linker refers to a linker which is capable of being split inresponse to the presence of a chemical (e.g., acid, base, oxidizingagent, reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilutenitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodiumdithionite (Na₂S₂O₄), or hydrazine (N₂H₄)). A chemically cleavablelinker is non-enzymatically cleavable. In embodiments, the cleavablelinker is cleaved by contacting the cleavable linker with a cleavingagent. In embodiments, the cleaving agent is a phosphine containingreagent (e.g., TCEP or THPP), sodium dithionite (Na₂S₂O₄), weak acid,hydrazine (N₂H₄), Pd(0), or light-irradiation (e.g., ultravioletradiation). In embodiments, cleaving includes removing. A “cleavablesite” or “scissile linkage” in the context of a polynucleotide is a sitewhich allows controlled cleavage of the polynucleotide strand (e.g., thelinker, the primer, or the polynucleotide) by chemical, enzymatic, orphotochemical means known in the art and described herein. A scissilesite may refer to the linkage of a nucleotide between two othernucleotides in a nucleotide strand (i.e., an internucleosidic linkage).In embodiments, the scissile linkage can be located at any positionwithin the one or more nucleic acid molecules, including at or near aterminal end (e.g., the 3′ end of an oligonucleotide) or in an interiorportion of the one or more nucleic acid molecules. In embodiments,conditions suitable for separating a scissile linkage include amodulating the pH and/or the temperature. In embodiments, a scissilesite can include at least one acid-labile linkage. For example, anacid-labile linkage may include a phosphoramidate linkage. Inembodiments, a phosphoramidate linkage can be hydrolysable under acidicconditions, including mild acidic conditions such as trifluoroaceticacid and a suitable temperature (e.g., 30° C.), or other conditionsknown in the art, for example Matthias Mag, et al Tetrahedron Letters,Volume 33, Issue 48, 1992, 7319-7322. In embodiments, the scissile sitecan include at least one photolabile internucleosidic linkage (e.g.,o-nitrobenzyl linkages, as described in Walker et al, J. Am. Chem. Soc.1988, 110, 21, 7170-7177), such as o-nitrobenzyloxymethyl orp-nitrobenzyloxymethyl group(s). In embodiments, the scissile siteincludes at least one uracil nucleobase. In embodiments, a uracilnucleobase can be cleaved with a uracil DNA glycosylase (UDG) orFormamidopyrimidine DNA Glycosylase Fpg. In embodiments, the scissilelinkage site includes a sequence-specific nicking site having anucleotide sequence that is recognized and nicked by a nickingendonuclease enzyme or a uracil DNA glycosylase.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site blast.ncbi.nlm.nih.gov/Blast.cgi or the like). Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the complement of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. As described below,the preferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length.

As used herein, the term “removable” group, e.g., a label or a blockinggroup or protecting group, is used in accordance with its plain andordinary meaning and refers to a chemical group that can be removed froma nucleotide analogue such that a DNA polymerase can extend the nucleicacid (e.g., a primer or extension product) by the incorporation of atleast one additional nucleotide. Removal may be by any suitable method,including enzymatic, chemical, or photolytic cleavage. Removal of aremovable group, e.g., a blocking group, does not require that theentire removable group be removed, only that a sufficient portion of itbe removed such that a DNA polymerase can extend a nucleic acid byincorporation of at least one additional nucleotide using a nucleotideor nucleotide analogue. In general, the conditions under which aremovable group is removed are compatible with a process employing theremovable group (e.g., an amplification process or sequencing process).

As used herein, the terms “reversible blocking groups” and “reversibleterminators” are used in accordance with their plain and ordinarymeanings and refer to a blocking moiety located, for example, at the 3′position of the nucleotide and may be a chemically cleavable moiety suchas an allyl group, an azidomethyl group or a methoxymethyl group, or maybe an enzymatically cleavable group such as a phosphate ester.Non-limiting examples of nucleotide blocking moieties are described inapplications WO 2004/018497, U.S. Pat. Nos. 7,057,026, 7,541,444, WO96/07669, U.S. Pat. Nos. 5,763,594, 5,808,045, 5,872,244 and 6,232,465the contents of which are incorporated herein by reference in theirentirety. The nucleotides may be labelled or unlabeled. They may bemodified with reversible terminators useful in methods provided hereinand may be 3′-O-blocked reversible or 3′-unblocked reversibleterminators. In nucleotides with 3-O-blocked reversible terminators, theblocking group —OR [reversible terminating (capping) group] is linked tothe oxygen atom of the 3-OH of the pentose, while the label is linked tothe base, which acts as a reporter and can be cleaved. The 3-O-blockedreversible terminators are known in the art, and may be, for instance, a3′-ONH₂ reversible terminator, a 3′-O-allyl reversible terminator, or a3-O-azidomethyl reversible terminator. In embodiments, the reversibleterminator moiety is

The term “allyl” as described herein refers to an unsubstitutedmethylene attached to a vinyl group (i.e., —CH═CH₂), having the formula

In embodiments, the reversible terminator moiety is

as described in U.S. Pat. No. 10,738,072, which is incorporated hereinby reference for all purposes. For example, a nucleotide including areversible terminator moiety may be represented by the formula:

where the nucleobase is adenine or adenine analogue, thymine or thymineanalogue, guanine or guanine analogue, or cytosine or cytosine analogue.

In some embodiments, a nucleic acid comprises a molecular identifier ora molecular barcode. As used herein, the term “molecular barcode” (whichmay be referred to as a “tag”, a “barcode”, a “molecular identifier”, an“identifier sequence” or a “unique molecular identifier” (UMI)) refersto any material (e.g., a nucleotide sequence, a nucleic acid moleculefeature) that is capable of distinguishing an individual molecule in alarge heterogeneous population of molecules. In embodiments, a barcodeis unique in a pool of barcodes that differ from one another insequence, or is uniquely associated with a particular samplepolynucleotide in a pool of sample polynucleotides. In embodiments,every barcode in a pool of adapters is unique, such that sequencingreads comprising the barcode can be identified as originating from asingle sample polynucleotide molecule on the basis of the barcode alone.In other embodiments, individual barcode sequences may be used more thanonce, but adapters comprising the duplicate barcodes are associated withdifferent sequences and/or in different combinations of barcodedadapters, such that sequence reads may still be uniquely distinguishedas originating from a single sample polynucleotide molecule on the basisof a barcode and adjacent sequence information (e.g., samplepolynucleotide sequence, and/or one or more adjacent barcodes). Inembodiments, barcodes are about or at least about 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 40, 50, 75 or more nucleotides in length. In embodiments,barcodes are shorter than 20, 15, 10, 9, 8, 7, 6, or 5 nucleotides inlength. In embodiments, barcodes are about 10 to about 50 nucleotides inlength, such as about 15 to about 40 or about 20 to about 30 nucleotidesin length. In a pool of different barcodes, barcodes may have the sameor different lengths. In general, barcodes are of sufficient length andinclude sequences that are sufficiently different to allow theidentification of sequencing reads that originate from the same samplepolynucleotide molecule. In embodiments, each barcode in a plurality ofbarcodes differs from every other barcode in the plurality by at leastthree nucleotide positions, such as at least 3, 4, 5, 6, 7, 8, 9, 10, ormore nucleotide positions. In some embodiments, substantially degeneratebarcodes may be known as random.

In some embodiments, the reaction conditions for a plurality ofinvasion-primer extension cycles includes incubation in a denaturant. Asused herein, the terms “denaturant” or plural “denaturants” are used inaccordance with their plain and ordinary meanings and refer to anadditive or condition that disrupts the base pairing between nucleotideswithin opposing strands of a double-stranded polynucleotide molecule.The term “denature” and its variants, when used in reference to anydouble-stranded polynucleotide molecule, or double-strandedpolynucleotide sequence, includes any process whereby the base pairingbetween nucleotides within opposing strands of the double-strandedmolecule, or double-stranded sequence, is disrupted. Typically,denaturation includes rendering at least some portion or region of twostrands of the double-stranded polynucleotide molecule or sequencesingle-stranded or partially single-stranded. In some embodiments,denaturation includes separation of at least some portion or region oftwo strands of the double-stranded polynucleotide molecule or sequencefrom each other. Typically, the denatured region or portion is thencapable of hybridizing to another polynucleotide molecule or sequence.Optionally, there can be “complete” or “total” denaturation of adouble-stranded polynucleotide molecule or sequence. Completedenaturation conditions are, for example, conditions that would resultin complete separation of a significant fraction (e.g., more than 10%,20%, 30%, 40% or 50%) of a large plurality of strands from theirextended and/or full-length complements. Typically, complete or totaldenaturation disrupts all of the base pairing between the nucleotides ofthe two strands with each other. Similarly, a nucleic acid sample isoptionally considered fully denatured when more than 80% or 90% ofindividual molecules of the sample lack any double-strandedness (or lackany hybridization to a complementary strand).

Alternatively, the double-stranded polynucleotide molecule or sequencecan be partially or incompletely denatured. A given nucleic acidmolecule can be considered partially denatured when a portion of atleast one strand of the nucleic acid remains hybridized to acomplementary strand, while another portion is in an unhybridized state(even if it is in the presence of a complementary sequence). Theunhybridized portion is optionally at least 5, 10, 15, 20, 50, or morenucleotides in length. The hybridized portion is optionally at least 5,10, 15, 20, 50, or more nucleotides in length. Partial denaturationincludes situations where some, but not all, of the nucleotides of onestrand or sequence, are based paired with some nucleotides of the otherstrand or sequence within a double-stranded polynucleotide. In someembodiments, at least 20% but less than 100% of the nucleotide residuesof one strand of the partially denatured polynucleotide (or sequence)are not base paired to nucleotide residues within the opposing strand.In embodiments, at least 50% of nucleotide residues within thedouble-stranded polynucleotide molecule (or double-strandedpolynucleotide sequence) are in single-stranded (or unhybridized) from,but less than 20% or 10% of the residues are double-stranded.

Optionally, a nucleic acid sample can be considered to be partiallydenatured when a substantial fraction of individual nucleic acidmolecules of the sample (e.g., above 20%, 30%, 50%, or 70%) are in apartially denatured state. Optionally less than a substantial amount ofindividual nucleic acid molecules in the sample are fully denatured,e.g., not more than 5%, 10%, 20%, 30% or 50% of the nucleic acidmolecules in the sample. Under exemplary conditions at least 50% of thenucleic acid molecules of the sample are partly denatured, but less than20% or 10% are fully denatured. In other situations, at least 30% of thenucleic acid molecules of the sample are partly denatured, but less than10% or 5% are fully denatured. Similarly, a nucleic acid sample can benon-denatured when a minority of individual nucleic acid molecules inthe sample are partially or completely denatured.

In an embodiment, partially denaturing conditions are achieved bymaintaining the duplexes as a suitable temperature range. For example,the nucleic acid is maintained at temperature sufficiently elevated toachieve some heat-denaturation (e.g., above 45° C., 50° C., 55° C., 60°C., 65° C., or 70° C.) but not high enough to achieve completeheat-denaturation (e.g., below 95° C. or 90° C. or 85° C. or 80° C. or75° C.). In an embodiment the nucleic acid is partially denatured usingsubstantially isothermal conditions. Alternatively, chemicaldenaturation can be accomplished by contacting the double-strandedpolynucleotide to be denatured with appropriate chemical denaturants,such as strong alkalis, strong acids, chaotropic agents, and the likeand can include, for example, NaOH, urea, or guanidine-containingcompounds. In some embodiments, partial or complete denaturation isachieved by exposure to chemical denaturants such as urea or formamide,with concentrations suitably adjusted, or using high or low pH (e.g., pHbetween 4-6 or 8-9). In embodiments, the denaturant is a bufferedsolution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol,formamide, glycerol, guanidine thiocyanate, 4-methylmorpholine 4-oxide(NMO), or a mixture thereof. In embodiments, the first denaturant is abuffered solution including about 0% to about 50% dimethyl sulfoxide(DMSO); about 0% to about 50% ethylene glycol; about 0% to about 20%formamide; or about 0 to about 3M betaine, or a mixture thereof. In anembodiment herein, partial denaturation and/or amplification, includingany one or more steps or methods described herein, can be achieved usinga recombinase and/or single-stranded binding protein.

In some embodiments, complete or partial denaturation of adouble-stranded polynucleotide sequence is accomplished by contactingthe double-stranded polynucleotide sequence using appropriate denaturingagents. For example, the double-stranded polynucleotide can be subjectedto heat-denaturation (also referred to interchangeably as thermaldenaturation) by raising the temperature to a point where the desiredlevel of denaturation is accomplished. In some embodiments, thermaldenaturation of a double-stranded polynucleotide, includes adjusting thetemperature to achieve complete separation of the two strands of thepolynucleotide, such that 90% or greater of the strands are insingle-stranded form across their entire length. A completely denatureddouble-stranded polynucleotide results in a separated first strand and asecond strand, each of which is a single-stranded polynucleotide. Insome embodiments, complete thermal denaturation of a polynucleotidemolecule (or polynucleotide sequence) is accomplished by exposing thepolynucleotide molecule (or sequence) to a temperature that is at least5° C., 10° C., 15° C., 20° C., 25° C., 30° C., 50° C., or 100° C., abovethe calculated or predict melting temperature (Tm) of the polynucleotidemolecule or sequence.

In some embodiments, complete or partial denaturation is accomplished bytreating the double-stranded polynucleotide sequence to be denaturedusing a denaturant mixture including an SSB protein (e.g., T4 gp32protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcuskodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobussolfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNABinding Protein (ET-SSB)), a strand-displacing polymerase (e.g., Bstlarge fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst 2.0polymerase, Bsu polymerase, SD polymerase, Vent exo-polymerase, Phi29polymerase, or a mutant thereof), and one or more crowding agents(poly(ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), bovine serumalbumin (BSA), dextran, Ficoll (e.g., Ficoll 70 or Ficoll 400),glycerol, or a combination thereof). In embodiments, the crowding agentis poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050,PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000),dextran sulfate, bovine pancreatic trypsin inhibitor (BPTI),ribonuclease A, lysozyme, β-lactoglobulin, hemoglobin, bovine serumalbumin (BSA), or poly(sodium 4-styrene sulfonate) (PSS). Inembodiments, the denaturant mixture including an SSB, astrand-displacing polymerase, and one or more crowding agents does notinclude a chemical denaturant (e.g., betaine, DMSO, ethylene glycol,formamide, guanidine thiocyanate, NMO, TMAC, or a mixture thereof).

In some embodiments, a nucleic acid comprises a label. As used herein,the term “label” or “labels” are used in accordance with their plain andordinary meanings and refer to molecules that can directly or indirectlyproduce or result in a detectable signal either by themselves or uponinteraction with another molecule. Non-limiting examples of detectablelabels include fluorescent dyes, biotin, digoxin, haptens, and epitopes.In general, a dye is a molecule, compound, or substance that can providean optically detectable signal, such as a colorimetric, luminescent,bioluminescent, chemiluminescent, phosphorescent, or fluorescent signal.In embodiments, the label is a dye. In embodiments, the dye is afluorescent dye. Non-limiting examples of dyes, some of which arecommercially available, include CF dyes (Biotium, Inc.), Alexa Fluordyes (Thermo Fisher), DyLight dyes (Thermo Fisher), Cy dyes (GEHealthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes(Anaspec, Inc.). In embodiments, a particular nucleotide type isassociated with a particular label, such that identifying the labelidentifies the nucleotide with which it is associated. In embodiments,the label is luciferin that reacts with luciferase to produce adetectable signal in response to one or more bases being incorporatedinto an elongated complementary strand, such as in pyrosequencing. Inembodiment, a nucleotide comprises a label (such as a dye). Inembodiments, the label is not associated with any particular nucleotide,but detection of the label identifies whether one or more nucleotideshaving a known identity were added during an extension step (such as inthe case of pyrosequencing).

In embodiments, the detectable label is a fluorescent dye. Inembodiments, the detectable label is a fluorescent dye capable ofexchanging energy with another fluorescent dye (e.g., fluorescenceresonance energy transfer (FRET) chromophores). Examples of detectableagents include imaging agents, including fluorescent and luminescentsubstances, including, but not limited to, a variety of organic orinorganic small molecules commonly referred to as “dyes,” “labels,” or“indicators.” Examples include fluorescein, rhodamine, acridine dyes,Alexa dyes, and cyanine dyes. In embodiments, the detectable moiety is afluorescent molecule (e.g., acridine dye, cyanine, dye, fluorine dye,oxazine dye, phenanthridine dye, or rhodamine dye). In embodiments, thedetectable moiety is a fluorescent molecule (e.g., acridine dye,cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, orrhodamine dye). In embodiments, the detectable moiety is a moiety of aderivative of one of the detectable moieties described immediatelyabove, wherein the derivative differs from one of the detectablemoieties immediately above by a modification resulting from theconjugation of the detectable moiety to a compound described herein.

The term “cyanine” or “cyanine moiety” as described herein refers to adetectable moiety containing two nitrogen groups separated by apolymethine chain. In embodiments, the cyanine moiety has 3 methinestructures (i.e., cyanine 3 or Cy3). In embodiments, the cyanine moietyhas 5 methine structures (i.e., cyanine 5 or Cy5). In embodiments, thecyanine moiety has 7 methine structures (i.e., cyanine 7 or Cy7).

As used herein, the term “DNA polymerase” and “nucleic acid polymerase”are used in accordance with their plain ordinary meanings and refer toenzymes capable of synthesizing nucleic acid molecules from nucleotides(e.g., deoxyribonucleotides). Typically, a DNA polymerase addsnucleotides to the 3′-end of a DNA strand, one nucleotide at a time. Inembodiments, the DNA polymerase is a Pol I DNA polymerase, Pol II DNApolymerase, Pol III DNA polymerase, Pol IV DNA polymerase, Pol V DNApolymerase, Pol β DNA polymerase, Pol μ DNA polymerase, Pol λ DNApolymerase, Pol σ DNA polymerase, Pol α DNA polymerase, Pol δ DNApolymerase, Pol ε DNA polymerase, Pol η DNA polymerase, Pol τ DNApolymerase, Pol κ DNA polymerase, Pol ζ DNA polymerase, Pol γ DNApolymerase, Pol θ DNA polymerase, Pol ν DNA polymerase, or athermophilic nucleic acid polymerase (e.g. Therminator γ, 9° Npolymerase (exo-), Therminator II, Therminator III, or Therminator IX).In embodiments, the DNA polymerase is a modified archaeal DNApolymerase. In embodiments, the polymerase is a reverse transcriptase.In embodiments, the polymerase is a mutant P. abyssi polymerase (e.g.,such as a mutant P. abyssi polymerase described in WO 2018/148723 or WO2020/056044). In embodiments, the polymerase is an enzyme described inUS 2021/0139884. For example, a polymerase catalyzes the addition of anext correct nucleotide to the 3′-OH group of the primer via aphosphodiester bond, thereby chemically incorporating the nucleotideinto the primer. Optionally, the polymerase used in the provided methodsis a processive polymerase. Optionally, the polymerase used in theprovided methods is a distributive polymerase.

As used herein, the term “thermophilic nucleic acid polymerase” refersto a family of DNA polymerases (e.g., 9° Nm) and mutants thereof derivedfrom the DNA polymerase originally isolated from the hyperthermophilicarchaea, Thermococcus sp. 9 degrees N-7, found in hydrothermal vents atthat latitude (East Pacific Rise) (Southworth M W, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member ofthe family B DNA polymerases. Site-directed mutagenesis of the 3′-5′ exomotif I (Asp-Ile-Glu or DIE) to ALA, AIE, EIE, EID or DIA yieldedpolymerase with no detectable 3′ exonuclease activity. Mutation toAsp-Ile-Asp (DID) resulted in reduction of 3′-5′ exonuclease specificactivity to <1% of wild type, while maintaining other properties of thepolymerase including its high strand displacement activity. The sequenceAIA (D141A, E143A) was chosen for reducing exonuclease. Subsequentmutagenesis of key amino acids results in an increased ability of theenzyme to incorporate dideoxynucleotides, ribonucleotides andacyclonucleotides (e.g., Therminator II enzyme from New England Biolabswith D141A/E143A/Y409V/A485L mutations); 3′-amino-dNTPs, 3′-azido-dNTPsand other 3′-modified nucleotides (e.g., NEB Therminator III DNAPolymerase with D141A/E143A/L408S/Y409A/P410V mutations, NEB TherminatorIX DNA polymerase), or γ-phosphate labeled nucleotides (e.g.,Therminator γ:D141A/E143A/W355A/L408W/R460A/Q461S/K464E/D480V/R484W/A485L). Typically,these enzymes do not have 5′-3′ exonuclease activity. Additionalinformation about thermophilic nucleic acid polymerases may be found in(Southworth M W, et al. PNAS. 1996; 93(11):5281-5285; Bergen K, et al.ChemBioChem. 2013; 14(9):1058-1062; Kumar S, et al. Scientific Reports.2012; 2:684; Fuller C W, et al. 2016; 113(19):5233-5238; Guo J, et al.Proceedings of the National Academy of Sciences of the United States ofAmerica. 2008; 105(27):9145-9150), which are incorporated herein intheir entirety for all purposes.

As used herein, the term “exonuclease activity” is used in accordancewith its ordinary meaning in the art, and refers to the removal of anucleotide from a nucleic acid by a DNA polymerase. For example, duringpolymerization, nucleotides are added to the 3′ end of the primerstrand. Occasionally a DNA polymerase incorporates an incorrectnucleotide to the 3′-OH terminus of the primer strand, wherein theincorrect nucleotide cannot form a hydrogen bond to the correspondingbase in the template strand. Such a nucleotide, added in error, isremoved from the primer as a result of the 3′ to 5′ exonuclease activityof the DNA polymerase. In embodiments, exonuclease activity may bereferred to as “proofreading.” When referring to 3′-5′ exonucleaseactivity, it is understood that the DNA polymerase facilitates ahydrolyzing reaction that breaks phosphodiester bonds at the 3′ end of apolynucleotide chain to excise the nucleotide. In embodiments, 3′-5′exonuclease activity refers to the successive removal of nucleotides insingle-stranded DNA in a 3′→5′ direction, releasing deoxyribonucleoside5′-monophosphates one after another. Methods for quantifying exonucleaseactivity are known in the art, see for example Southworth et al, PNASVol 93, 8281-8285 (1996). In embodiments, 5′-3′ exonuclease activityrefers to the successive removal of nucleotides in double-stranded DNAin a 5′→3′ direction. In embodiments, the 5′-3′ exonuclease is lambdaexonuclease. For example, lambda exonuclease catalyzes the removal of 5′mononucleotides from duplex DNA, with a preference for 5′ phosphorylateddouble-stranded DNA. In other embodiments, the 5′-3′ exonuclease is E.coli DNA Polymerase I.

As used herein, the term “incorporating” or “chemically incorporating,”when used in reference to a primer and a nucleotide, refers to theprocess of joining the nucleotide to the primer or extension productthereof by formation of a phosphodiester bond.

As used herein, the term “selective” or “selectivity” or the like of acompound refers to the compound's ability to discriminate betweenmolecular targets. When used in the context of sequencing, such as in“selectively sequencing,” this term refers to sequencing one or moretarget polynucleotides from an original starting population ofpolynucleotides, and not sequencing non-target polynucleotides from thestarting population. Typically, selectively sequencing one or moretarget polynucleotides involves differentially manipulating the targetpolynucleotides based on known sequence. For example, targetpolynucleotides may be hybridized to a probe oligonucleotide that may belabeled (such as with a member of a binding pair) or bound to a surface.In embodiments, hybridizing a target polynucleotide to a probeoligonucleotide includes the step of displacing one strand of adouble-stranded nucleic acid. Probe-hybridized target polynucleotidesmay then be separated from non-hybridized polynucleotides, such as byremoving probe-bound polynucleotides from the starting population or bywashing away polynucleotides that are not bound to a probe. The resultis a selected subset of the starting population of polynucleotides,which is then subjected to sequencing, thereby selectively sequencingthe one or more target polynucleotides.

As used herein, the terms “specific”, “specifically”, “specificity”, orthe like of a compound refers to the agent's ability to cause aparticular action, such as binding, to a particular molecular targetwith minimal or no action to other proteins in the cell.

As used herein, the terms “bind” and “bound” are used in accordance withtheir plain and ordinary meanings and refer to an association betweenatoms or molecules. The association can be direct or indirect. Forexample, bound atoms or molecules may be directly bound to one another,e.g., by a covalent bond or non-covalent bond (e.g., electrostaticinteractions (e.g., ionic bond, hydrogen bond, halogen bond), van derWaals interactions (e.g., dipole-dipole, dipole-induced dipole, Londondispersion), ring stacking (pi effects), hydrophobic interactions andthe like). As a further example, two molecules may be bound indirectlyto one another by way of direct binding to one or more intermediatemolecules, thereby forming a complex.

As used herein, the term “rolling circle amplification (RCA)” refers toa nucleic acid amplification reaction that amplifies a circular nucleicacid template (e.g., single-stranded DNA circles) via a rolling circlemechanism. Rolling circle amplification reaction is initiated by thehybridization of a primer to a circular, often single-stranded, nucleicacid template. The nucleic acid polymerase then extends the primer thatis hybridized to the circular nucleic acid template by continuouslyprogressing around the circular nucleic acid template to replicate thesequence of the nucleic acid template over and over again (rollingcircle mechanism). The rolling circle amplification typically producesconcatemers comprising tandem repeat units of the circular nucleic acidtemplate sequence. The rolling circle amplification may be a linear RCA(LRCA), exhibiting linear amplification kinetics (e.g., RCA using asingle specific primer), or may be an exponential RCA (ERCA) exhibitingexponential amplification kinetics. Rolling circle amplification mayalso be performed using multiple primers (multiply primed rolling circleamplification or MPRCA) leading to hyper-branched concatemers. Forexample, in a double-primed RCA, one primer may be complementary, as inthe linear RCA, to the circular nucleic acid template, whereas the othermay be complementary to the tandem repeat unit nucleic acid sequences ofthe RCA product. Consequently, the double-primed RCA may proceed as achain reaction with exponential (geometric) amplification kineticsfeaturing a ramifying cascade of multiple-hybridization,primer-extension, and strand-displacement events involving both theprimers. This often generates a discrete set of concatemeric,double-stranded nucleic acid amplification products. The rolling circleamplification may be performed in-vitro under isothermal conditionsusing a suitable nucleic acid polymerase such as Phi29 DNA polymerase.RCA may be performed by using any of the DNA polymerases that are knownin the art (e.g., a Phi29 DNA polymerase, a Bst DNA polymerase, or SDpolymerase).

As used herein, the terms “sequencing”, “sequence determination”,“determining a nucleotide sequence”, and the like include determinationof a partial or complete sequence information, including theidentification, ordering, or locations of the nucleotides that comprisethe polynucleotide being sequenced, and inclusive of the physicalprocesses for generating such sequence information. That is, the termincludes sequence comparisons, consensus sequence determination, contigassembly, fingerprinting, and like levels of information about a targetpolynucleotide, as well as the express identification and ordering ofnucleotides in a target polynucleotide. The term also includes thedetermination of the identification, ordering, and locations of one,two, or three of the four types of nucleotides within a targetpolynucleotide. In some embodiments, a sequencing process describedherein comprises contacting a template and an annealed primer with asuitable polymerase under conditions suitable for polymerase extensionand/or sequencing. The sequencing methods are preferably carried outwith the target polynucleotide arrayed on a solid substrate. Multipletarget polynucleotides can be immobilized on the solid support throughlinker molecules, or can be attached to particles, e.g., microspheres,which can also be attached to a solid substrate. In embodiments, thesolid substrate is in the form of a chip, a bead, a well, a capillarytube, a slide, a wafer, a filter, a fiber, a porous media, or a column.In embodiments, the solid substrate is gold, quartz, silica, plastic,glass, diamond, silver, metal, or polypropylene. In embodiments, thesolid substrate is porous.

As used herein, the term “sequencing cycle” is used in accordance withits plain and ordinary meaning and refers to incorporating one or morenucleotides (e.g., nucleotide analogues) to the 3′ end of apolynucleotide with a polymerase, and detecting the one or morenucleotides incorporated. In embodiments, one nucleotide (e.g., amodified nucleotide) is incorporated per sequencing cycle. Thesequencing may be accomplished by, for example, sequencing by synthesis,pyrosequencing, and the like. In embodiments, a sequencing cycleincludes extending a complementary polynucleotide by incorporating afirst nucleotide using a polymerase, wherein the polynucleotide ishybridized to a template nucleic acid, detecting the first nucleotide,and identifying the first nucleotide. An “extension strand” is formed asthe one or more nucleotides are incorporated into a complementarypolynucleotide hybridized to a template nucleic acid. The extensionstrand is complementary to the template nucleic acid. In embodiments, tobegin a sequencing cycle, one or more differently labeled nucleotidesand a DNA polymerase can be introduced. Following nucleotide addition,signals produced (e.g., via excitation and emission of a detectablelabel) can be detected to determine the identity of the incorporatednucleotide (based on the labels on the nucleotides). Reagents can thenbe added to remove the 3′ reversible terminator and to remove labelsfrom each incorporated base. Reagents, enzymes, and other substances canbe removed between steps by washing. Cycles may include repeating thesesteps, and the sequence of each cluster is read over the multiplerepetitions.

As used herein, the term “sequencing reaction mixture” is used inaccordance with its plain and ordinary meaning and refers to an aqueousmixture that contains the reagents necessary to allow a nucleotide ornucleotide analogue to be added (i.e., incorporated) to a DNA strand bya DNA polymerase. As used herein, the term “invasion-reaction mixture”is used in accordance with its plain and ordinary meaning and refers toan aqueous mixture that contains the reagents necessary to allow anucleotide or nucleotide analogue to be added to a DNA strand by a DNApolymerase that extends the invasion primer.

As used herein, the term “extension” or “elongation” is used inaccordance with their plain and ordinary meanings and refer to synthesisby a polymerase of a new polynucleotide strand (i.e., an “extensionstrand”) complementary to a template strand by adding free nucleotides(e.g., dNTPs) from a reaction mixture that are complementary to thetemplate in a 5′-to-3′ direction, including condensing a 5′-phosphategroup of a dNTPs with a 3′-hydroxy group at the end of the nascent(elongating) DNA strand.

As used herein, the term “sequencing read” is used in accordance withits plain and ordinary meaning and refers to an inferred sequence ofnucleotide bases (or nucleotide base probabilities) corresponding to allor part of a single polynucleotide fragment. A sequencing read mayinclude 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or morenucleotide bases. In embodiments, a sequencing read includes reading abarcode and a template nucleotide sequence. In embodiments, a sequencingread includes reading a template nucleotide sequence. In embodiments, asequencing read includes reading a barcode and not a template nucleotidesequence. In embodiments, a sequencing read includes a computationallyderived string corresponding to the detected label. The sequence readsare optionally stored in an appropriate data structure for furtherevaluation. In embodiments, a first sequencing reaction can generate afirst sequencing read. The first sequencing read can provide thesequence of a first region of the polynucleotide fragment. Inembodiments, a second sequencing primer can initiate sequencing at asecond location on the nucleic acid template. The second location can bedistinct from the first location. In some cases, a 3′ terminalnucleotide of the second primer can hybridize to a location that is morethan 5 nucleotides away from a binding site of a 3′ terminal nucleotideof the first primer. The second sequencing reaction can generate asecond sequencing read. The second sequencing read can provide thesequence of a second region of the nucleic acid template which isdistinct from the first region of the nucleic acid template. In someembodiments, the nucleic acid template is optionally subjected to one ormore additional rounds of sequencing using additional sequencingprimers, thereby generating additional sequencing reads.

The term “multiplexing” as used herein refers to an analytical method inwhich the presence and/or amount of multiple targets, e.g., multiplenucleic acid target sequences, can be assayed simultaneously by usingthe methods and devices as described herein, each of which has at leastone different detection characteristic, e.g., fluorescencecharacteristic (for example excitation wavelength, emission wavelength,emission intensity, FWHM (full width at half maximum peak height), orfluorescence lifetime) or a unique nucleic acid or protein sequencecharacteristic.

As used herein, the term “hybridize” or “specifically hybridize” refersto a process where two complementary nucleic acid strands anneal to eachother under appropriately stringent conditions. Hybridizations aretypically and preferably conducted with oligonucleotides. The terms“annealing” and “hybridization” are used interchangeably to mean theformation of a stable duplex. In some embodiments, one portion of anucleic acid hybridizes to itself, such as in the formation of a hairpinstructure. The propensity for hybridization between nucleic acidsdepends on the temperature and ionic strength of their milieu, thelength of the nucleic acids and the degree of complementarity. Theeffect of these parameters on hybridization is described in, forexample, Sambrook J., Fritsch E. F., Maniatis T., Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, New York (1989).As used herein, hybridization of a primer, or of a DNA extensionproduct, respectively, is extendable by creation of a phosphodiesterbond with an available nucleotide or nucleotide analogue capable offorming a phosphodiester bond, therewith. For example, hybridization canbe performed at a temperature ranging from 15° C. to 95° C. In someembodiments, the hybridization is performed at a temperature of about20° C., about 25° C., about 30° C., about 35° C., about 40° C., about45° C., about 50° C., about 55° C., about 60° C., about 65° C., about70° C., about 75° C., about 80° C., about 85° C., about 90° C., or about95° C. In other embodiments, the stringency of the hybridization can befurther altered by the addition or removal of components of the bufferedsolution. Those skilled in the art understand how to estimate and adjustthe stringency of hybridization conditions such that sequences having atleast a desired level of complementarity will stably hybridize, whilethose having lower complementarity will not. As used herein, the term“stringent condition” refers to condition(s) under which apolynucleotide probe or primer will hybridize preferentially to itstarget sequence, and to a lesser extent to, or not at all to, othersequences. In some embodiments nucleic acids, or portions thereof, thatare configured to specifically hybridize are often about 80% or more,81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% ormore, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, 99% or more or 100% complementary to each other overa contiguous portion of nucleic acid sequence. A specific hybridizationdiscriminates over non-specific hybridization interactions (e.g., twonucleic acids that a not configured to specifically hybridize, e.g., twonucleic acids that are 80% or less, 70% or less, 60% or less or 50% orless complementary) by about 2-fold or more, often about 10-fold ormore, and sometimes about 100-fold or more, 1000-fold or more,10,000-fold or more, 100,000-fold or more, or 1,000,000-fold or more.Two nucleic acid strands (e.g., two single-stranded polynucleotides)that are hybridized to each other can form a duplex which comprises adouble-stranded portion of nucleic acid.

A nucleic acid can be amplified by a suitable method. The term“amplified” as used herein refers to subjecting a target nucleic acid ina sample to a process that linearly or exponentially generates ampliconnucleic acids having the same or substantially the same (e.g.,substantially identical) nucleotide sequence as the target nucleic acid,or segment thereof, and/or a complement thereof. In some embodiments anamplification reaction comprises a suitable thermal stable polymerase.Thermal stable polymerases are known in the art and are stable forprolonged periods of time, at temperature greater than 80° C. whencompared to common polymerases found in most mammals. In certainembodiments the term “amplified” refers to a method that comprises apolymerase chain reaction (PCR). Conditions conducive to amplification(i.e., amplification conditions) are known and often comprise at least asuitable polymerase, a suitable template, a suitable primer or set ofprimers, suitable nucleotides (e.g., dNTPs), a suitable buffer, andapplication of suitable annealing, hybridization and/or extension timesand temperatures. In certain embodiments an amplified product (e.g., anamplicon) can contain one or more additional and/or differentnucleotides than the template sequence, or portion thereof, from whichthe amplicon was generated (e.g., a primer can contain “extra”nucleotides (such as a 5′ portion that does not hybridize to thetemplate), or one or more mismatched bases within a hybridizing portionof the primer).

A nucleic acid can be amplified by a thermocycling method or by anisothermal amplification method. In some embodiments, a rolling circleamplification method is used. In some embodiments, amplification takesplace on a solid support (e.g., within a flow cell) where a nucleicacid, nucleic acid library or portion thereof is immobilized. In certainsequencing methods, a nucleic acid library is added to a flow cell andimmobilized by hybridization to anchors under suitable conditions. Thistype of nucleic acid amplification is often referred to as solid phaseamplification. In some embodiments of solid phase amplification, all ora portion of the amplified products are synthesized by an extensioninitiating from an immobilized primer. Solid phase amplificationreactions are analogous to standard solution phase amplifications exceptthat at least one of the amplification oligonucleotides (e.g., primers)is immobilized on a solid support.

In some embodiments solid phase amplification comprises a nucleic acidamplification reaction comprising only one species of oligonucleotideprimer immobilized to a surface or substrate. In certain embodimentssolid phase amplification comprises a plurality of different immobilizedoligonucleotide primer species. In some embodiments solid phaseamplification may comprise a nucleic acid amplification reactioncomprising one species of oligonucleotide primer immobilized on a solidsurface and a second different oligonucleotide primer species insolution. Multiple different species of immobilized or solution-basedprimers can be used. Non-limiting examples of solid phase nucleic acidamplification reactions include interfacial amplification, bridgeamplification, emulsion PCR, WildFire amplification (e.g., US patentpublication US20130012399), the like or combinations thereof.

Provided herein are methods and compositions for analyzing a sample(e.g., sequencing nucleic acids within a sample). A sample (e.g., asample comprising nucleic acid) can be obtained from a suitable subject.A sample can be isolated or obtained directly from a subject or partthereof. In some embodiments, a sample is obtained indirectly from anindividual or medical professional. A sample can be any specimen that isisolated or obtained from a subject or part thereof. A sample can be anyspecimen that is isolated or obtained from multiple subjects.Non-limiting examples of specimens include fluid or tissue from asubject, including, without limitation, blood or a blood product (e.g.,serum, plasma, platelets, buffy coats, or the like), umbilical cordblood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinalfluid, lavage fluid (e.g., lung, gastric, peritoneal, ductal, ear,arthroscopic), a biopsy sample, celocentesis sample, cells (blood cells,lymphocytes, placental cells, stem cells, bone marrow derived cells,embryo or fetal cells) or parts thereof (e.g., mitochondrial, nucleus,extracts, or the like), urine, feces, sputum, saliva, nasal mucous,prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat,breast milk, breast fluid, the like or combinations thereof. A fluid ortissue sample from which nucleic acid is extracted may be acellular(e.g., cell-free). Non-limiting examples of tissues include organtissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder,reproductive organs, intestine, colon, spleen, brain, the like or partsthereof), epithelial tissue, hair, hair follicles, ducts, canals, bone,eye, nose, mouth, throat, ear, nails, the like, parts thereof orcombinations thereof. A sample may comprise cells or tissues that arenormal, healthy, diseased (e.g., infected), and/or cancerous (e.g.,cancer cells). A sample obtained from a subject may comprise cells orcellular material (e.g., nucleic acids) of multiple organisms (e.g.,virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasitenucleic acid).

In some embodiments, a sample comprises nucleic acid, or fragmentsthereof. A sample can comprise nucleic acids obtained from one or moresubjects. In some embodiments a sample comprises nucleic acid obtainedfrom a single subject. In some embodiments, a sample comprises a mixtureof nucleic acids. A mixture of nucleic acids can comprise two or morenucleic acid species having different nucleotide sequences, differentfragment lengths, different origins (e.g., genomic origins, cell ortissue origins, subject origins, the like or combinations thereof), orcombinations thereof. A sample may comprise synthetic nucleic acid.

A subject can be any living or non-living organism, including but notlimited to a human, non-human animal, plant, bacterium, fungus, virus orprotist. A subject may be any age (e.g., an embryo, a fetus, infant,child, adult). A subject can be of any sex (e.g., male, female, orcombination thereof). A subject may be pregnant. In some embodiments, asubject is a mammal. In some embodiments, a subject is a human subject.A subject can be a patient (e.g., a human patient). In some embodimentsa subject is suspected of having a genetic variation or a disease orcondition associated with a genetic variation.

The terms “bioconjugate group,” “bioconjugate reactive moiety,” and“bioconjugate reactive group” refer to a chemical moiety whichparticipates in a reaction to form a bioconjugate linker (e.g., covalentlinker). Non-limiting examples of bioconjugate groups include —NH₂,—COOH, —COOCH₃, —N-hydroxysuccinimide, -maleimide,

In embodiments, the bioconjugate reactive group may be protected (e.g.,with a protecting group). In embodiments, the bioconjugate reactivemoiety is

or —NH₂. Additional examples of bioconjugate reactive groups and theresulting bioconjugate reactive linkers may be found in the BioconjugateTable below:

Bioconjugate reactive Bioconjugate reactive group 1 (e.g., group 2(e.g., Resulting electrophilic nucleophilic Bioconjugate bioconjugatereactive bioconjugate reactive reactive moiety) moiety) linker activatedesters amines/anilines carboxamides acrylamides thiols thioethers acylazides amines/anilines carboxamides acyl halides amines/anilinescarboxamides acyl halides alcohols/phenols esters acyl nitrilesalcohols/phenols esters acyl nitriles amines/anilines carboxamidesaldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolsthiophenols aryl halides amines aryl amines aziridines thiols thioethersboronates glycols boronate esters carbodiimides carboxylic acidsN-acylureas or diazoalkanes carboxylic acids anhydrides epoxides thiolsesters haloacetamides thiols thioethers haloplatinate amino thioethershaloplatinate heterocycle platinum complex haloplatinate thiol platinumcomplex halotriazines amines/anilines platinum complex halotriazinesalcohols/phenols aminotriazines halotriazines thiols triazinyl ethersimido esters amines/anilines triazinyl thioethers isocyanatesamines/anilines amidines isocyanates alcohols/phenols ureasisothiocyanates amines/anilines urethanes maleimides thiols thioureasphosphoramidites alcohols thioethers silyl halides alcohols phosphiteesters sulfonate esters amines/anilines silyl ethers sulfonate estersthiols alkyl amines sulfonate esters carboxylic acids thioetherssulfonate esters alcohols esters sulfonyl halides amines/anilines etherssulfonyl halides phenols/alcohols sulfonamides sulfonate esters

As used herein, the term “bioconjugate” or “bioconjugate linker” refersto the resulting association between atoms or molecules of bioconjugatereactive groups. The association can be direct or indirect. For example,a conjugate between a first bioconjugate reactive group (e.g., —NH₂,—COOH, —N-hydroxysuccinimide, or -maleimide) and a second bioconjugatereactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine,amine sidechain containing amino acid, or carboxylate) provided hereincan be direct, e.g., by covalent bond or linker (e.g., a first linker ofsecond linker), or indirect, e.g., by non-covalent bond (e.g.,electrostatic interactions (e.g., ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g., dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). In embodiments, bioconjugates orbioconjugate linkers are formed using bioconjugate chemistry (i.e., theassociation of two bioconjugate reactive groups) including, but notlimited to nucleophilic substitutions (e.g., reactions of amines andalcohols with acyl halides, active esters), electrophilic substitutions(e.g., enamine reactions) and additions to carbon-carbon andcarbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alderaddition). These and other useful reactions are discussed in, forexample, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons,New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, SanDiego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances inChemistry Series, Vol. 198, American Chemical Society, Washington, D.C.,1982. In embodiments, the first bioconjugate reactive group (e.g.,maleimide moiety) is covalently attached to the second bioconjugatereactive group (e.g., a sulfhydryl). In embodiments, the firstbioconjugate reactive group (e.g., haloacetyl moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g., pyridylmoiety) is covalently attached to the second bioconjugate reactive group(e.g., a sulfhydryl). In embodiments, the first bioconjugate reactivegroup (e.g., —N-hydroxysuccinimide moiety) is covalently attached to thesecond bioconjugate reactive group (e.g., an amine). In embodiments, thefirst bioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g.,-sulfo-N-hydroxysuccinimide moiety) is covalently attached to the secondbioconjugate reactive group (e.g., an amine). In embodiments, the firstbioconjugate reactive group (e.g., —COOH) is covalently attached to thesecond bioconjugate reactive group

thereby forming a bioconjugate

In embodiments, the first bioconjugate reactive group (e.g., —NH₂) iscovalently attached to the second bioconjugate reactive group

thereby forming a bioconjugate

In embodiments, the first bioconjugate reactive group (e.g., a couplingreagent) is covalently attached to the second bioconjugate reactivegroup

thereby forming a bioconjugate

The bioconjugate reactive groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of theconjugate described herein. Alternatively, a reactive functional groupcan be protected from participating in the crosslinking reaction by thepresence of a protecting group. In embodiments, the bioconjugatecomprises a molecular entity derived from the reaction of an unsaturatedbond, such as a maleimide, and a sulfhydryl group.

Useful bioconjugate reactive groups used for bioconjugate chemistriesherein include, for example: (a) carboxyl groups and various derivativesthereof including, but not limited to, N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b)hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.; (c) haloalkyl groups wherein the halide can be later displacedwith a nucleophilic group such as, for example, an amine, a carboxylateanion, thiol anion, carbanion, or an alkoxide ion, thereby resulting inthe covalent attachment of a new group at the site of the halogen atom;(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido or maleimide groups; (e)aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition; (f) sulfonyl halide groupsfor subsequent reaction with amines, for example, to form sulfonamides;(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold, or react withmaleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine),which can be, for example, acylated, alkylated or oxidized; (i) alkenes,which can undergo, for example, cycloadditions, acylation, Michaeladdition, etc.; (j) epoxides, which can react with, for example, aminesand hydroxyl compounds; (k) phosphoramidites and other standardfunctional groups useful in nucleic acid synthesis; (l) metal siliconoxide bonding; (m) metal bonding to reactive phosphorus groups (e.g.,phosphines) to form, for example, phosphate diester bonds; (n) azidescoupled to alkynes using copper catalyzed cycloaddition click chemistry;(o) biotin conjugate can react with avidin or streptavidin to form aavidin-biotin complex or streptavidin-biotin complex.

The term “covalent linker” is used in accordance with its ordinarymeaning and refers to a divalent moiety which connects at least twomoieties to form a molecule. The term “non-covalent linker” is used inaccordance with its ordinary meaning and refers to a divalent moietywhich includes at least two molecules that are not covalently linked toeach other but are capable of interacting with each other via anon-covalent bond (e.g., electrostatic interactions (e.g., ionic bond,hydrogen bond, halogen bond) or van der Waals interactions (e.g.,dipole-dipole, dipole-induced dipole, London dispersion). Inembodiments, the non-covalent linker is the result of two molecules thatare not covalently linked to each other that interact with each othervia a non-covalent bond.

The term “adapter” as used herein refers to any oligonucleotide that canbe ligated to a nucleic acid molecule, thereby generating nucleic acidproducts that can be sequenced on a sequencing platform (e.g., anIllumina or Singular Genomics G4™ sequencing platform). In embodiments,adapters include two reverse complementary oligonucleotides forming adouble-stranded structure. In embodiments, an adapter includes twooligonucleotides that are complementary at one portion and mismatched atanother portion, forming a Y-shaped or fork-shaped adapter that isdouble stranded at the complementary portion and has two overhangs atthe mismatched portion. Since Y-shaped adapters have a complementary,double-stranded region, they can be considered a special form ofdouble-stranded adapters. When this disclosure contrasts Y-shapedadapters and double stranded adapters, the term “double-strandedadapter” or “blunt-ended” is used to refer to an adapter having twostrands that are fully complementary, substantially (e.g., more than 90%or 95%) complementary, or partially complementary. In embodiments,adapters include sequences that bind to sequencing primers. Inembodiments, adapters include sequences that bind to immobilizedoligonucleotides (e.g., P7 and P5 sequences) or reverse complementsthereof. In embodiments, the adapter is substantially non-complementaryto the 3′ end or the 5′ end of any target polynucleotide present in thesample. In embodiments, the adapter can include a sequence that issubstantially identical, or substantially complementary, to at least aportion of a primer, for example a universal primer. In embodiments, theadapter can include an index sequence (also referred to as barcode ortag) to assist with downstream error correction, identification orsequencing. In some embodiments, an adapter is hairpin adapter. In someembodiments, a hairpin adapter comprises a single nucleic acid strandcomprising a stem-loop structure. In some embodiments, a hairpin adaptercomprises a nucleic acid having a 5′-end, a 5′-portion, a loop, a3′-portion and a 3′-end (e.g., arranged in a 5′ to 3′ orientation). Insome embodiments, the 5′ portion of a hairpin adapter is annealed and/orhybridized to the 3′ portion of the hairpin adapter, thereby forming astem portion of the hairpin adapter. In some embodiments, the 5′ portionof a hairpin adapter is substantially complementary to the 3′ portion ofthe hairpin adapter. In certain embodiments, a hairpin adapter comprisesa stem portion (i.e., stem) and a loop, wherein the stem portion issubstantially double stranded thereby forming a duplex. In someembodiments, the loop of a hairpin adapter comprises a nucleic acidstrand that is not complementary (e.g., not substantially complementary)to itself or to any other portion of the hairpin adapter. In someembodiments, a method herein comprises ligating a first adapter to afirst end of a double stranded nucleic acid, and ligating a secondadapter to a second end of a double stranded nucleic acid. In someembodiments, the first adapter and the second adapter are different. Forexample, in certain embodiments, the first adapter and the secondadapter may comprise different nucleic acid sequences or differentstructures. In some embodiments, the first adapter is a Y-adapter andthe second adapter is a hairpin adapter. In some embodiments, the firstadapter is a hairpin adapter and a second adapter is a hairpin adapter.In certain embodiments, the first adapter and the second adapter maycomprise different primer binding sites, different structures, and/ordifferent capture sequences (e.g., a sequence complementary to a capturenucleic acid). In some embodiments, some, all or substantially all ofthe nucleic acid sequence of a first adapter and a second adapter arethe same. In some embodiments, some, all or substantially all of thenucleic acid sequence of a first adapter and a second adapter aresubstantially different.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly indicates otherwise, between the upper and lowerlimit of that range, and any other stated or unstated intervening valuein, or smaller range of values within, that stated range is encompassedwithin the invention. The upper and lower limits of any such smallerrange (within a more broadly recited range) may independently beincluded in the smaller ranges, or as particular values themselves, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

“Synthetic” agents refer to non-naturally occurring agents, such asenzymes or nucleotides derived or constructed using man-made techniques.Synthetic DNA polymerases refer to non-naturally occurring DNApolymerases such as those constructed by synthetic methods, mutatedparent DNA polymerases such as truncated DNA polymerases and fusion DNApolymerases (e.g. U.S. Pat. No. 7,541,170). Variants of the parent DNApolymerase have been engineered by mutating residues using site-directedor random mutagenesis methods known in the art. In embodiments, themutations are in any of Motifs I-VI. The variant is expressed in anexpression system such as E. coli by methods known in the art.

The methods and kits of the present disclosure may be applied, mutatismutandis, to the sequencing of RNA, or to determining the identity of aribonucleotide.

“GC bias” describes the relationship between GC content and readcoverage across a genome. For example, a genomic region of a higher GCcontent tends to have more (or less) sequencing reads covering thatregion. As described herein, GC bias can be introduced duringamplification of library, cluster amplification, and/or the sequencingreactions.

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,        —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,        —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,        —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —O CHI₂, —OCHF₂,        —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted alkyl        (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted        cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆        cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8        membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or        5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g.,        C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted heteroaryl        (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl,        or 5 to 6 membered heteroaryl), and    -   (B) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g.,        C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),        heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6        membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),        aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), heteroaryl (e.g.,        5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to        6 membered heteroaryl), substituted with at least one        substituent selected from:        -   (i) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,            —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,            —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,            —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,            —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,            —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F,            —N₃, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or            C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8            membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4            membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈            cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),            unsubstituted heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), unsubstituted aryl (e.g.,            C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted            heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9            membered heteroaryl, or 5 to 6 membered heteroaryl), and        -   (ii) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),            heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6            membered heteroalkyl, or 2 to 4 membered heteroalkyl),            cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or            C₅-C₆ cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), aryl (e.g., C₆-C₁₀ aryl, C₁₀            aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered            heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered            heteroaryl), substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,                —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN,                —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,                —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,                —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃,                —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,                —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted                alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),                unsubstituted heteroalkyl (e.g., 2 to 8 membered                heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4                membered heteroalkyl), unsubstituted cycloalkyl (e.g.,                C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆                cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to                8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                unsubstituted aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or                phenyl), or unsubstituted heteroaryl (e.g., 5 to 10                membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to                6 membered heteroaryl), and            -   (b) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄                alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl,                2 to 6 membered heteroalkyl, or 2 to 4 membered                heteroalkyl), cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), heterocycloalkyl                (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl),                heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9                membered heteroaryl, or 5 to 6 membered heteroaryl),                substituted with at least one substituent selected from:                oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,                —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,                —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —S O₂NH₂,                —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,                —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,                —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,                —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted alkyl (e.g.,                C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted                heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6                membered heteroalkyl, or 2 to 4 membered heteroalkyl),                unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), unsubstituted                heterocycloalkyl (e.g., 3 to 8 membered                heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5                to 6 membered heterocycloalkyl), unsubstituted aryl                (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or                unsubstituted heteroaryl (e.g., 5 to 10 membered                heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6                membered heteroaryl).

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted phenyl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 6membered heteroaryl.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals. The alkyl may include a designated number ofcarbons (e.g., C₁-C₁₀ means one to ten carbons). In embodiments, thealkyl is fully saturated. In embodiments, the alkyl is monounsaturated.In embodiments, the alkyl is polyunsaturated. Alkyl is an uncyclizedchain. Examples of saturated hydrocarbon radicals include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. An alkoxy is an alkylattached to the remainder of the molecule via an oxygen linker (—O—). Analkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynylmoiety. An alkyl moiety may be fully saturated. An alkenyl may includemore than one double bond and/or one or more triple bonds in addition tothe one or more double bonds. An alkynyl may include more than onetriple bond and/or one or more double bonds in addition to the one ormore triple bonds. An alkenyl includes one or more double bonds. Analkynyl includes one or more triple bonds.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen andsulfur atoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P)may be placed at any interior position of the heteroalkyl group or atthe position at which the alkyl group is attached to the remainder ofthe molecule. Heteroalkyl is an uncyclized chain. Examples include, butare not limited to: —CH₂—CH₂O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—S—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CHO—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. A heteroalkyl moiety mayinclude one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moietymay include two optionally different heteroatoms (e.g., O, N, S, Si, orP). A heteroalkyl moiety may include three optionally differentheteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includefour optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include five optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8optionally different heteroatoms (e.g., O, N, S, Si, or P). The term“heteroalkenyl,” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one doublebond. A heteroalkenyl may optionally include more than one double bondand/or one or more triple bonds in additional to the one or more doublebonds. The term “heteroalkynyl,” by itself or in combination withanother term, means, unless otherwise stated, a heteroalkyl including atleast one triple bond. A heteroalkynyl may optionally include more thanone triple bond and/or one or more double bonds in additional to the oneor more triple bonds. In embodiments, the heteroalkyl is fullysaturated. In embodiments, the heteroalkyl is monounsaturated. Inembodiments, the heteroalkyl is polyunsaturated.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. The term“heteroalkenylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from a heteroalkene.The term “heteroalkynylene” by itself or as part of another substituent,means, unless otherwise stated, a divalent radical derived from aheteroalkyne. In embodiments, the heteroalkylene is fully saturated. Inembodiments, the heteroalkylene is monounsaturated. In embodiments, theheteroalkylene is polyunsaturated. A heteroalkenylene includes one ormore double bonds. A heteroalkynylene includes one or more triple bonds.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

II. Compositions, Substrates, & Kits

In an aspect is a substrate (e.g., a solid support) including a firstpolynucleotide attached to the substrate; a second polynucleotideattached to the substrate, wherein the second polynucleotide includes acomplementary sequence to the first polynucleotide; and a thirdpolynucleotide (alternatively referred to herein as an invasion primeror extended invasion primer) hybridized to the second polynucleotide. Inembodiments, the substrate further includes a plurality of immobilizedoligonucleotides (e.g., immobilized primers, such as immobilized forwardand immobilized reverse primers) attached to the substrate via a linker.In embodiments, the first and second polynucleotides are covalentlyattached to the substrate. In embodiments, the 5′ end of the first andsecond polynucleotides contains a functional group that serves to tetherthe first and second polynucleotides to the substrate (e.g., abioconjugate linker). Non-limiting examples of covalent attachmentinclude amine-modified polynucleotides reacting with epoxy orisothiocyanate groups on the substrate, succinylated polynucleotidesreacting with aminophenyl or aminopropyl functional groups on thesubstrate, dibenzocycloctyne-modified polynucleotides reacting withazide functional groups on the substrate (or vice versa),trans-cyclooctyne-modified polynucleotides reacting with tetrazine ormethyl tetrazine groups on the substrate (or vice versa), disulfidemodified polynucleotides reacting with mercapto-functional groups on thesubstrate, amine-functionalized polynucleotides reacting with carboxylicacid groups on the core via1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)chemistry, thiol-modified polynucleotides attaching to a substrate via adisulfide bond or maleimide linkage, alkyne-modified polynucleotidesattaching to a substrate via copper-catalyzed click reactions to azidefunctional groups on the substrate, and acrydite-modifiedpolynucleotides polymerizing with free acrylic acid monomers on thesubstrate to form polyacrylamide or reacting with thiol groups on thesubstrate. In embodiments, the primer is attached to the substratepolymer through electrostatic binding. For example, the negativelycharged phosphate backbone of the primer may be bound electrostaticallyto positively charged monomers in the substrate. In embodiments, thethird polynucleotide is not covalently attached to the substrate.

In embodiments, the substrate includes a plurality of firstpolynucleotides attached to a solid support; a plurality of secondpolynucleotides attached to a solid support; and a plurality of thirdpolynucleotides hybridized to each of the second polynucleotides. It isunderstood that when referring to first, second, and thirdpolynucleotides it is in reference to a class of polynucleotide types.For example, the polynucleotides of the first polynucleotides aresubstantially similar to each other insomuch as they containsubstantially identical sequences.

In embodiments, the third polynucleotide, which may also be referred toas the invasion primer and is interchangeable with the thirdpolynucleotide, includes locked nucleic acids (LNAs), Bis-locked nucleicacids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridgednucleic acids (BNAs), 2′-O-methyl RNA:DNA chimeric nucleic acids, minorgroove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modifiedpyrimidine nucleic acids, peptide nucleic acids (PNAs), or combinationsthereof. In embodiments, the third polynucleotide includes Bis-lockednucleic acids (bisLNAs). In embodiments, the third polynucleotideincludes twisted intercalating nucleic acids (TINAs). In embodiments,the third polynucleotide includes bridged nucleic acids (BNAs). Inembodiments, the third polynucleotide includes 2′-O-methyl RNA:DNAchimeric nucleic acids. In embodiments, the third polynucleotideincludes minor groove binder (MGB) nucleic acids. In embodiments, thethird polynucleotide includes morpholino nucleic acids. Morpholinonucleic acids are synthetic nucleotides that have standard nucleic acidbases (e.g., adenine, guanine, cytosine, and thymine) wherein thosebases are bound to methylenemorpholine rings linked throughphosphorodiamidate groups instead of phosphates. Morpholino nucleicacids may be referred to as phosphorodiamidate morpholino oligomers(PMOs). In embodiments, the third polynucleotide includes C5-modifiedpyrimidine nucleic acids. In embodiments, the third polynucleotideincludes peptide nucleic acids (PNAs). In embodiments, the thirdpolynucleotide includes from 5′ to 3′ a plurality of syntheticnucleotides (e.g., LNAs) followed by a plurality (e.g., 2 to 5)canonical or native nucleotides (e.g., dNTPs). In embodiments, the thirdpolynucleotide comprises one or more (e.g., 2 to 5) deoxyuracilnucleobases (dU). In embodiments, the one or more dU nucleobases are ator near the 3′ end of the third polynucleotide (e.g., within 5nucleotides of the 3′ end). In embodiments, the third polynucleotideincludes from 5′ to 3′ a plurality (e.g., 2 to 5) of phosphorothioatenucleic acids, followed by a plurality of synthetic nucleotides (e.g.,LNAs), and subsequently followed by a plurality (e.g., 2 to 5) ofcanonical nucleobases. In some embodiments, the third polynucleotideincludes a plurality of canonical nucleobases, wherein the canonicalnucleobases terminate (i.e., at the 3′ end) with a deoxyuracilnucleobase (dU).

In embodiments, the third polynucleotide includes a plurality of LNAsinterspersed throughout the polynucleotide. In embodiments, the thirdpolynucleotide includes a plurality of consecutive LNAs (e.g., 2 to 5LNAs, 5 to 7 LNAs, or 7 to 10 LNAs) throughout the polynucleotide. Inembodiments, the entire composition of the third polynucleotide includesless than 70%, less than 60%, less than 50%, less than 40%, less than30%, less than 20%, less than 10%, or less than 5% of LNAs. Inembodiments, the entire composition of the third polynucleotide includesup to about 70%, up to about 60%, up to about 50%, up to about 40%, upto about 30%, up to about 20%, up to about 10%, or up to about 5% ofLNAs. In embodiments, the entire composition of the third polynucleotideincludes more than 60%, more than 50%, more than 40%, more than 30%,more than 20%, more than 10%, or more than 5% of LNAs. In embodiments,the entire composition of the third polynucleotide includes about 5% toabout 10%, about 10% to about 20%, about 20% to about 30%, about 30% toabout 40%, about 40% to about 50%, about 50% to about 60%, or about 60%to about 70% of LNAs. In embodiments, the entire composition of thethird polynucleotide includes about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, or about 70% of LNAs. In embodiments, theentire composition of the third polynucleotide includes about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% ofcanonical dNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes less than 95%, less than 90%, less than 80%,less than 70%, less than 60%, less than 50%, less than 40%, or less than30% of canonical dNTPs. In embodiments, the entire composition of thethird polynucleotide includes up to about 95%, up to about 90%, up toabout 80%, up to about 70%, up to about 60%, up to about 50%, up toabout 40%, or up to about 30% of canonical dNTPs. In embodiments, theentire composition of the third polynucleotide includes more than 90%,more than 80%, more than 70%, more than 60%, more than 50%, more than40%, or more than 30% of canonical dNTPs.

In embodiments, the entire composition of the third polynucleotideincludes about 70% of LNAs and about 30% of canonical dNTPs. Inembodiments, the entire composition of the third polynucleotide includesabout 65% of LNAs and about 35% of canonical dNTPs. In embodiments, theentire composition of the third polynucleotide includes about 60% ofLNAs and about 40% of canonical dNTPs. In embodiments, the entirecomposition of the third polynucleotide includes about 55% of LNAs andabout 45% of canonical dNTPs. In embodiments, the entire composition ofthe third polynucleotide includes about 50% of LNAs and about 50% ofcanonical dNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 45% of LNAs and about 55% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 40% of LNAs and about 60% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 35% of LNAs and about 65% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 30% of LNAs and about 70% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 25% of LNAs and about 75% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 20% of LNAs and about 80% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 15% of LNAs and about 85% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 10% of LNAs and about 90% of canonicaldNTPs. In embodiments, the entire composition of the thirdpolynucleotide includes about 5% of LNAs and about 95% of canonicaldNTPs.

In embodiments, the third polynucleotide includes one or more dTnucleobases that are replaced with dU nucleobases. In embodiments, thethird polynucleotide includes a plurality of dT nucleobases that arereplaced with dU nucleobases. In embodiments, the third polynucleotideincludes all dT nucleobases replaced with dU nucleobases. Inembodiments, the third polynucleotide includes dU nucleobases and LNAnucleotides. In embodiments, the third polynucleotide includes dUnucleobases and LNA nucleotides, wherein the LNA nucleotides are notadjacent to the dU nucleobases.

In embodiments, the third polynucleotide includes a homologousrecombination complex including a recombinase bound thereto. Inembodiments, the homologous recombination complex further includes aloading factor, a single-stranded binding (SSB) protein, or both.

In embodiments, the substrate includes a silica surface including apolymer coating. In embodiments, the substrate is silica or quartz, suchas a microscope slide, having a surface that is uniformly silanized.This may be accomplished using conventional protocols, such as thosedescribed in Beattie et al (1995), Molecular Biotechnology, 4: 213. Sucha surface is readily treated to permit end-attachment ofoligonucleotides (e.g., forward and reverse primers) prior toamplification. In embodiments the substrate surface further includes apolymer coating, which contains functional groups capable ofimmobilizing primers. In some embodiments, the substrate includes apatterned surface suitable for immobilization of primers in an orderedpattern. A patterned surface refers to an arrangement of differentregions in or on an exposed layer of a substrate. For example, one ormore of the regions can be features where one or more primers arepresent. The features can be separated by interstitial regions wherecapture primers are not present. In some embodiments, the pattern can bean x-y format of features that are in rows and columns. In someembodiments, the pattern can be a repeating arrangement of featuresand/or interstitial regions. In some embodiments, the pattern can be arandom arrangement of features and/or interstitial regions. In someembodiments, the primers are randomly distributed upon the substrate. Insome embodiments, the primers are distributed on a patterned surface.

In embodiments, the first polynucleotide is immobilized on the substratevia a first linker and the second polynucleotide is immobilized to thesubstrate via a second linker. The linkers may also include spacernucleotides. Including spacer nucleotides in the linker puts thepolynucleotide in an environment having a greater resemblance to freesolution. This can be beneficial, for example, in enzyme-mediatedreactions such as sequencing-by-synthesis. It is believed that suchreactions suffer less steric hindrance issues that can occur when thepolynucleotide is directly attached to the solid support or is attachedthrough a very short linker (e.g., a linker comprising about 1 to 3carbon atoms). Spacer nucleotides form part of the polynucleotide but donot participate in any reaction carried out on or with thepolynucleotide (e.g. a hybridization or amplification reaction). Inembodiments, the spacer nucleotides include 1 to 20 nucleotides. Inembodiments, the linker includes 10 spacer nucleotides. In embodiments,the linker includes 12 spacer nucleotides. In embodiments, the linkerincludes 15 spacer nucleotides. It is preferred to use polyT spacers,although other nucleotides and combinations thereof can be used. Inembodiments, the linker includes 10, 11, 12, 13, 14, or 15 T spacernucleotides. In embodiments, the linker includes 12 T spacernucleotides. Spacer nucleotides are typically included at the 5′ ends ofpolynucleotides which are attached to a suitable support. Attachment canbe achieved via a phosphorothioate present at the 5′ end of thepolynucleotide, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, orany other bioconjugate reactive moiety. The linker may be acarbon-containing chain such as those of formula —(CH₂)n- wherein “n” isfrom 1 to about 1000. However, a variety of other linkers may be used solong as the linkers are stable under conditions used in DNA sequencing.In embodiments, the linker includes polyethylene glycol (PEG) having ageneral formula of —(CH₂—CH₂—O)m-, wherein m is from about 1 to 500, 1to 100, or 1 to 12.

In embodiments, the linker, or the immobilized oligonucleotides (e.g.,primers) include a cleavable site. In embodiments, a cleavable site is alocation which allows controlled cleavage of the immobilizedpolynucleotide strand (e.g., the linker, the primer, or thepolynucleotide) by chemical, enzymatic or photochemical means. Inembodiments, the cleavable site includes one or more deoxyuracilnucleobases (dUs).

Any suitable enzymatic, chemical, or photochemical cleavage reaction maybe used to cleave the cleavable site. The cleavage reaction may resultin removal of a part or the whole of the strand being cleaved. Suitablecleavage means include, for example, restriction enzyme digestion, inwhich case the cleavable site is an appropriate restriction site for theenzyme which directs cleavage of one or both strands of a duplextemplate; RNase digestion or chemical cleavage of a bond between adeoxyribonucleotide and a ribonucleotide, in which case the cleavablesite may include one or more ribonucleotides; chemical reduction of adisulfide linkage with a reducing agent (e.g., THPP or TCEP), in whichcase the cleavable site should include an appropriate disulfide linkage;chemical cleavage of a diol linkage with periodate, in which case thecleavable site should include a diol linkage; generation of an abasicsite and subsequent hydrolysis, etc. In embodiments, the cleavable siteis included in the surface immobilized primer (e.g., within thepolynucleotide sequence of the primer). In embodiments, the linker, theprimer, or the first or second polynucleotide includes a diol linkagewhich permits cleavage by treatment with periodate (e.g., sodiumperiodate). It will be appreciated that more than one diol can beincluded at the cleavable site. One or more diol units may beincorporated into a polynucleotide using standard methods for automatedchemical DNA synthesis. Polynucleotide primers including one or morediol linkers can be conveniently prepared by chemical synthesis. Thediol linker is cleaved by treatment with any substance which promotescleavage of the diol (e.g., a diol-cleaving agent). In embodiments, thediol-cleaving agent is periodate, e.g., aqueous sodium periodate(NaIO₄). Following treatment with the diol-cleaving agent (e.g.,periodate) to cleave the diol, the cleaved product may be treated with a“capping agent” in order to neutralize reactive species generated in thecleavage reaction. Suitable capping agents for this purpose includeamines, e.g., ethanolamine or propanolamine. In embodiments, cleavagemay be accomplished by using a modified nucleotide as the cleavable site(e.g., uracil, 8oxoG, 5-mC, 5-hmC) that is removed or nicked via acorresponding DNA glycosylase, endonuclease, or combination thereof.

In embodiments, each of the plurality of immobilized oligonucleotides(e.g., immobilized primers) is about 5 to about 25 nucleotides inlength. In embodiments, each of the plurality of immobilizedoligonucleotides (e.g., immobilized primers) is about 10 to about 40nucleotides in length. In embodiments, each of the plurality ofimmobilized oligonucleotides (e.g., immobilized primers) is about 5 toabout 100 nucleotides in length. In embodiments, each of the pluralityof immobilized oligonucleotides (e.g., immobilized primers) is about 20to 200 nucleotides in length. In embodiments, each of the plurality ofimmobilized oligonucleotides (e.g., immobilized primers) about or atleast about 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 50 ormore nucleotides in length. In embodiments, one or more immobilizedoligonucleotides include blocking groups at their 3′ ends that preventpolymerase extension. A blocking moiety prevents formation of a covalentbond between the 3′ hydroxyl moiety of the nucleotide and the 5′phosphate of another nucleotide. In embodiments, the 3′ modification isa 3′-phosphate modification, including a 3′ phosphate moiety, which isremoved by a PNK enzyme or a phosphatase enzyme. Alternatively, abasicsite cleavage with certain endonucleases (e.g., Endo IV) results in a3′-OH at the cleavable site from the 3′-diesterase activity.

In embodiments, the immobilized oligonucleotides includes one or morephosphorothioate nucleic acids. In embodiments, the immobilizedoligonucleotides includes a plurality of phosphorothioate nucleic acids.In embodiments, about or at least about 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or about 100% of the nucleotides in the immobilizedoligonucleotides are phosphorothioate nucleic acids. In embodiments,most of the nucleotides in the immobilized oligonucleotides arephosphorothioate nucleic acids. In embodiments, all of the nucleotidesin the immobilized oligonucleotides are phosphorothioate nucleic acids.In embodiments, none of the nucleotides in the immobilizedoligonucleotides are phosphorothioate nucleic acids. In embodiments, the5′ end of the immobilized oligonucleotide includes one or morephosphorothioate nucleic acids. In embodiments, the 5′ end of theimmobilized oligonucleotide includes between one and fivephosphorothioate nucleic acids.

In embodiments, the first and second polynucleotides are each attachedto the solid support (i.e., immobilized on the surface of a solidsupport). The polynucleotide molecules can be fixed to surface by avariety of techniques, including covalent attachment and non-covalentattachment. In embodiments, the polynucleotides are confined to an areaof a discrete region (referred to as a cluster). The discrete regionsmay have defined locations in a regular array, which may correspond to arectilinear pattern, circular pattern, hexagonal pattern, or the like. Aregular array of such regions is advantageous for detection and dataanalysis of signals collected from the arrays during an analysis. Thesediscrete regions are separated by interstitial regions. As used herein,the term “interstitial region” refers to an area in a substrate or on asurface that separates other areas of the substrate or surface. Forexample, an interstitial region can separate one concave feature of anarray from another concave feature of the array. The two regions thatare separated from each other can be discrete, lacking contact with eachother. In another example, an interstitial region can separate a firstportion of a feature from a second portion of a feature. In embodimentsthe interstitial region is continuous whereas the features are discrete,for example, as is the case for an array of wells in an otherwisecontinuous surface. The separation provided by an interstitial regioncan be partial or full separation. Interstitial regions will typicallyhave a surface material that differs from the surface material of thefeatures on the surface. For example, features of an array can havepolynucleotides that exceeds the amount or concentration present at theinterstitial regions. In some embodiments the polynucleotides and/orprimers may not be present at the interstitial regions. In embodiments,at least two different primers are attached to the solid support (e.g.,a forward and a reverse primer), which facilitates generating multipleamplification products from the first extension product or a complementthereof.

In embodiments of the methods and compositions provided herein, theclusters have a mean or median separation from one another of about0.5-5 μm. In embodiments, the mean or median separation is about 0.1-10microns, 0.25-5 microns, 0.5-2 microns, 1 micron, or a number or a rangebetween any two of these values. In embodiments, the mean or medianseparation is about or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4., 4.5, 4.6, 4.7, 4.8, 4.9,5.0 μm or a number or a range between any two of these values. Inembodiments, the mean or median separation is about 0.1-10 microns. Inembodiments, the mean or median separation is about 0.25-5 microns. Inembodiments, the mean or median separation is about 0.5-2 microns. Inembodiments, the mean or median separation is about or at least about0.1 μm. In embodiments, the mean or median separation is about or atleast about 0.25 μm. In embodiments, the mean or median separation isabout or at least about 0.5 μm. In embodiments, the mean or medianseparation is about or at least about 1.0 μm. In embodiments, the meanor median separation is about or at least about 2.0 μm. In embodiments,the mean or median separation is about or at least about 5.0 μm. Inembodiments, the mean or median separation is about or at least about 10μm. The mean or median separation may be measured center-to-center(i.e., the center of one cluster to the center of a second cluster). Inembodiments of the methods provided herein, the amplicon clusters have amean or median separation (measured center-to-center) from one anotherof about 0.5-5 μm. The mean or median separation may be measurededge-to-edge (i.e., the edge of one amplicon cluster to the edge of asecond amplicon cluster). In embodiments of the methods provided herein,the amplicon clusters have a mean or median separation (measurededge-to-edge) from one another of about 0.2-5 μm.

In embodiments of the methods provided herein, the amplicon clustershave a mean or median diameter of about 100-2000 nm, or about 200-1000nm. In embodiments, the mean or median diameter is about 100-3000nanometers, about 500-2500 nanometers, about 1000-2000 nanometers, or anumber or a range between any two of these values. In embodiments, themean or median diameter is about or at most about 100, 200, 300, 400,500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, 2000 nanometers or a number or a rangebetween any two of these values. In embodiments, the mean or mediandiameter is about 100-3,000 nanometers. In embodiments, the mean ormedian diameter is about 100-2,000 nanometers. In embodiments, the meanor median diameter is about 500-2500 nanometers. In embodiments, themean or median diameter is about 200-1000 nanometers. In embodiments,the mean or median diameter is about 1,000-2,000 nanometers. Inembodiments, the mean or median diameter is about or at most about 100nanometers. In embodiments, the mean or median diameter is about or atmost about 200 nanometers. In embodiments, the mean or median diameteris about or at most about 500 nanometers. In embodiments, the mean ormedian diameter is about or at most about 400 nanometers. Inembodiments, the mean or median diameter is about or at most about 500nanometers. In embodiments, the mean or median diameter is about or atmost about 600 nanometers. In embodiments, the mean or median diameteris about or at most about 700 nanometers. In embodiments, the mean ormedian diameter is about or at most about 1,000 nanometers. Inembodiments, the mean or median diameter is about or at most about 2,000nanometers. In embodiments, the mean or median diameter is about or atmost about 2,500 nanometers. In embodiments, the mean or median diameteris about or at most about 3,000 nanometers.

In embodiments of the methods provided herein, each amplicon cluster(e.g., an amplicon cluster having a mean or median diameter of about100-2000 nm, or about 200-1000 nm) includes about or at least about 100,500, 1,000, 2,500, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000,35,000, 40,000, 45,000, or 50,000 dsDNA molecules. In embodiments, eachamplicon cluster includes about 100 dsDNA molecules. In embodiments,each amplicon cluster includes about 500 dsDNA molecules. Inembodiments, each amplicon cluster includes about 1000 dsDNA molecules.In embodiments, each amplicon cluster includes about 500 dsDNAmolecules. In embodiments, each amplicon cluster includes about 1,000dsDNA molecules. In embodiments, each amplicon cluster includes about2,500 dsDNA molecules. In embodiments, each amplicon cluster includesabout 5,000 dsDNA molecules. In embodiments, each amplicon clusterincludes about 10,000 dsDNA molecules. In embodiments, each ampliconcluster includes about 20,000 dsDNA molecules. In embodiments, eachamplicon cluster includes about 30,000 dsDNA molecules. In embodiments,each amplicon cluster includes about 40,000 dsDNA molecules. Inembodiments, each amplicon cluster includes about 50,000 dsDNAmolecules. In embodiments, each amplicon cluster includes more thanabout 50,000 dsDNA molecules.

In embodiments, the substrate is a particle. In embodiments, thesubstrate is a multiwell container. In embodiments, the substrate is apolymer coated particle or polymer coated planar support. Inembodiments, the substrate includes a polymer. In embodiments, theparticle includes polymerized units of polyacrylamide (AAm),poly-N-isopropylacrylamide, poly N-isopropylpolyacrylamide, sulfobetaineacrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholineacrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetainemethacrylate (CBMA), phosphorylcholine methacrylate (PCMA), polyethyleneglycol acrylate, methacrylate, polyethylene glycol(PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, glicydyl methacrylate (GMA),hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate (HEA),hydroxypropylmethacrylate (HPMA), polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethylmethacrylate (IEM), or a copolymer thereof. In embodiments, the particleshell includes polymerized units of polyacrylamide (AAm), glicydylmethacrylate (GMA), polyethylene glycol methacrylate (PEGMA),polyethylene glycol methacrylate (PEGMA), isocyanatoethyl methacrylate(IEM), or a copolymer thereof. In embodiments, the particle includespolymerized units of polyethylene glycol methacrylate (PEGMA) andglicydyl methacrylate (GMA). In embodiments, the particle includespolymerized units of polyethylene glycol methacrylate (PEGMA) andisocyanatoethyl methacrylate (IEM). In embodiments, the particleincludes polymerized units of 3-azido-2-hydroxypropyl methacrylate,2-azido-3-hydroxypropyl methacrylate,2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate,3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, theparticle includes polymerized units of 3-azido-2-hydroxypropylmethacrylate, 2-azido-3-hydroxypropyl methacrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments,the particle includes polymerized units of 3-azido-2-hydroxypropylmethacrylate. In embodiments, the particle includes polymerized units of3-azido-2-hydroxypropyl methacrylate 2-azido-3-hydroxypropylmethacrylate. In embodiments, the particle includes polymerized units of3-azido-2-hydroxypropyl methacrylate2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate.

In an aspect is a kit, wherein the kit includes the substrate asdescribed herein. Generally, the kit includes one or more containersproviding a composition and one or more additional reagents (e.g., abuffer suitable for polynucleotide extension). The kit may also includea template nucleic acid (DNA and/or RNA), one or more primerpolynucleotides, nucleoside triphosphates (including, e.g.,deoxyribonucleotides, ribonucleotides, labeled nucleotides, and/ormodified nucleotides), buffers, salts, and/or labels (e.g.,fluorophores).

In embodiments, the kit includes a sequencing polymerase, and one ormore amplification polymerases. In embodiments, the sequencingpolymerase is capable of incorporating modified nucleotides. Inembodiments, the polymerase is a DNA polymerase. In embodiments, the DNApolymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNApolymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol β DNApolymerase, Pol μ DNA polymerase, Pol λ DNA polymerase, Pol σ DNApolymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNApolymerase, Pol η DNA polymerase, Pol τ DNA polymerase, Pol κ DNApolymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNApolymerase, Pol ν DNA polymerase, or a thermophilic nucleic acidpolymerase (e.g., Therminator γ, 9° N polymerase (exo-), Therminator II,Therminator III, or Therminator IX). In embodiments, the DNA polymeraseis a thermophilic nucleic acid polymerase. In embodiments, the DNApolymerase is a modified archaeal DNA polymerase. In embodiments, thepolymerase is a reverse transcriptase. In embodiments, the polymerase isa mutant P. abyssi polymerase (e.g., such as a mutant P. abyssipolymerase described in WO 2018/148723 or WO 2020/056044, each of whichare incorporated herein by reference for all purposes). In embodiments,the kit includes a strand-displacing polymerase. In embodiments, the kitincludes a strand-displacing polymerase, such as a phi29 polymerase,phi29 mutant polymerase or a thermostable phi29 mutant polymerase.

In embodiments, the kit includes a buffered solution. Typically, thebuffered solutions contemplated herein are made from a weak acid and itsconjugate base or a weak base and its conjugate acid. For example,sodium acetate and acetic acid are buffer agents that can be used toform an acetate buffer. Other examples of buffer agents that can be usedto make buffered solutions include, but are not limited to, Tris,bicine, tricine, HEPES, TES, MOPS, MOPSO and PIPES. Additionally, otherbuffer agents that can be used in enzyme reactions, hybridizationreactions, and detection reactions are known in the art. In embodiments,the buffered solution can include Tris. With respect to the embodimentsdescribed herein, the pH of the buffered solution can be modulated topermit any of the described reactions. In some embodiments, the bufferedsolution can have a pH greater than pH 7.0, greater than pH 7.5, greaterthan pH 8.0, greater than pH 8.5, greater than pH 9.0, greater than pH9.5, greater than pH 10, greater than pH 10.5, greater than pH 11.0, orgreater than pH 11.5. In other embodiments, the buffered solution canhave a pH ranging, for example, from about pH 6 to about pH 9, fromabout pH 8 to about pH 10, or from about pH 7 to about pH 9. Inembodiments, the buffered solution can comprise one or more divalentcations. Examples of divalent cations can include, but are not limitedto, Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺. In embodiments, the buffered solutioncan contain one or more divalent cations at a concentration sufficientto permit hybridization of a nucleic acid.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay, etc.) from one locationto another. For example, kits include one or more enclosures (e.g.,boxes) containing the relevant reaction reagents and/or supportingmaterials. As used herein, the term “fragmented kit” refers to adelivery system comprising two or more separate containers that eachcontain a subportion of the total kit components. The containers may bedelivered to the intended recipient together or separately. For example,a first container may contain an enzyme for use in an assay, while asecond container contains oligonucleotides. In contrast, a “combinedkit” refers to a delivery system containing all of the components of areaction assay in a single container (e.g., in a single box housing eachof the desired components). The term “kit” includes both fragmented andcombined kits.

In an aspect is provided a polynucleotide (e.g., an invasion primer). Inembodiments, the polynucleotide includes a plurality of LNA nucleotides;one or more cleavable sites, wherein the one or more cleavable sitespartition the invasion primer into two or more regions; and a pluralityof native nucleotides.

In an aspect is provided a polynucleotide (e.g., an invasion primer). Inembodiments, the polynucleotide includes a plurality of LNA nucleotides;one or more dU nucleobases, wherein the one or more dU nucleobasespartition the invasion primer into two or more regions; and a pluralityof native nucleotides.

In embodiments, the polynucleotide is 20 to 40 nucleotides in length. Inembodiments, the polynucleotide is about 10 to 100 nucleotides inlength. In embodiments, the polynucleotide is about 15 to about 75nucleotides in length. In embodiments, the polynucleotide is about 25 toabout 75 nucleotides in length. In embodiments, the polynucleotide isabout 15 to about 50 nucleotides in length. In embodiments, thepolynucleotide is about 10 to about 20 nucleotides in length. Inembodiments, the polynucleotide is about 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or about 20 nucleotides in length. In embodiments, thepolynucleotide is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about30 nucleotides in length. In embodiments, the polynucleotide is about30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 nucleotides inlength. In embodiments, the polynucleotide is about 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or about 50 nucleotides in length. In embodiments,the polynucleotide is greater than 30 nucleotides in length. Inembodiments, the polynucleotide is greater than 40 nucleotides inlength. In embodiments, the polynucleotide is greater than 50nucleotides in length. In embodiments, the polynucleotide is no lessthan 20 nucleotides. In embodiments, the polynucleotide is about 15 toabout 35 nucleotides in length. In embodiments, the polynucleotide isabout 25 to about 35 nucleotides, wherein 12 to 18 nucleotides are LNAnucleotides. In embodiments, the polynucleotide is about 25 to about 35nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. Inembodiments, the polynucleotide is about 30 to about 35 nucleotides,wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, thepolynucleotide is 30, 31, 32, or 33 nucleotides, wherein 14 to 16nucleotides are LNA nucleotides.

In embodiments, the calculated or predicted melting temperature (Tm) ofthe polynucleotide is about 70° C. to about 95° C. In embodiments, thecalculated or predicted melting temperature (Tm) of the polynucleotideis about 80° C. to about 95° C. In embodiments, the calculated orpredicted melting temperature (Tm) of the polynucleotide is about 85° C.to about 95° C. In embodiments, the calculated or predicted meltingtemperature (Tm) of the polynucleotide is about 85° C. to about 90° C.In embodiments, the plurality of LNA nucleotides are interspersedthroughout the polynucleotide.

In embodiments, the one or more dU nucleobases partition thepolynucleotide into two or more regions of nucleotides (e.g., a firstplurality of consecutive nucleotides and a second plurality ofconsecutive nucleotides are separated by the one or more dUnucleobases). In embodiments each of the two or more regions ofconsecutive nucleotides are each about 3 to about 10 nucleotides inlength, or about 3 to about 15 nucleotides in length. In embodimentseach of the two or more regions of consecutive nucleotides are eachabout 3 to about 10 nucleotides in length. In embodiments each of thetwo or more regions of consecutive nucleotides are each about 3 to about15 nucleotides in length. In embodiments each of the two or more regionsof consecutive nucleotides are each at least about 3, 5, 7, 10, 13, or15 nucleotides in length. In embodiments, each of the two or moreregions of consecutive nucleotides is greater than about 15 nucleotidesin length. In embodiments, the calculated or predicted meltingtemperature (Tm) of each of the two or more regions of consecutivenucleotides is about 50° C. to about 75° C. In embodiments, thecalculated or predicted melting temperature (Tm) of each of the two ormore regions of consecutive nucleotides is about 60° C. to about 75° C.In embodiments, the calculated or predicted melting temperature (Tm) ofeach of the two or more regions of consecutive nucleotides is about 50°C. to about 65° C. In embodiments, the calculated or predicted meltingtemperature (Tm) of each of the two or more regions of consecutivenucleotides is less than about 75° C. In embodiments, the calculated orpredicted melting temperature (Tm) of each of the two or more regions ofconsecutive nucleotides is less than about 65° C. In embodiments, thecalculated or predicted melting temperature (Tm) of each of the two ormore regions of consecutive nucleotides is less than about 60° C.

III. Methods

In an aspect is provided a method of sequencing two or more regions of adouble-stranded polynucleotide including a first strand hybridized to asecond strand, wherein the first strand and second strand are bothattached to a solid support. In embodiments, the method includes: i)hybridizing an invasion primer to the second strand and extending theinvasion primer with a polymerase, thereby generating an invasionstrand; ii) hybridizing a sequencing primer to the first strand; iii)incorporating one or more nucleotides into the sequencing primer with apolymerase to create an extension strand; and iv) detecting the one ormore incorporated nucleotides so as to identify each incorporatednucleotide in the extension strand, thereby sequencing the first strandof the double-stranded polynucleotide. In embodiments, the methodfurther includes removing the first strand, removing the invasionstrand, or both removing the first strand and removing the invasionstrand. In embodiments, the method further includes removing theinvasion strand and hybridizing a second invasion primer to the firststrand and extending the second invasion primer with a polymerase,thereby generating a second invasion strand.

In embodiments, the method includes nicking and/or cleaving the invasionstrand to generate a 3′ end and incorporating one or more nucleotidesinto the 3′ end of the invasion primer with a polymerase to create anextension strand; and detecting the one or more incorporated nucleotidesso as to identify each incorporated nucleotide in said extension strand.

In an aspect is provided a method of forming a plurality ofsingle-stranded polynucleotides attached to a solid support. Inembodiments, the method includes: contacting a plurality ofdouble-stranded polynucleotides including a first strand hybridized to asecond strand with a plurality of invasion primers, wherein the firststrand and the second strand are attached to the solid support;hybridizing one or more invasion primers to the second strand; andextending one or more invasion primers hybridized to the second strandwith a polymerase to generate one or more invasion strands, displacingthe first strand, thereby forming a plurality of single-strandedpolynucleotides attached to the solid support. In embodiments, themethod further includes sequencing the single-stranded polynucleotides.In embodiments, the method further includes removing the invasion strandand sequencing the second strand.

In embodiments, the solid support includes about 100, 500, 1000, 5000,10000, or more dsDNA molecules in a 2 μm² area. In embodiments, thesolid support includes about 1,000 to about 10,000 dsDNA molecules in a2 μm² area. In embodiments, the solid support includes about 1,000 toabout 10,000 dsDNA molecules in a 0.5 μm diameter feature. Inembodiments, the solid support includes about 1,000 to about 50,000dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameterfeature. In embodiments, the solid support includes about 10,000 toabout 50,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nmdiameter feature. In embodiments, the solid support includes about20,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800, 900, or1,000 nm diameter feature. In embodiments, the solid support includesabout 30,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800,900, or 1,000 nm diameter feature. As used herein, a feature may be awells, pits, channels, ridges, raised regions, pegs, or posts on a solidsupport. Each feature includes a colony and refers to a discrete site ona solid support that includes a plurality of immobilizedpolynucleotides.

In embodiments, removing the invasion strand includes digesting (i.e.,cleaving internal phosphodiester bonds of a polynucleotide) all orportions thereof of the invasion strand using an exonuclease enzyme.Exonucleases can be active on ssDNA and/or dsDNA, initiate from the 5′end and/or the 3′ end of polynucleotides, and can also act on RNApolynucleotides. In embodiments, the exonuclease enzyme is a DNAspecific exonuclease. In embodiments, the exonuclease catalyzes theremoval of nucleotides from linear, and/or nicked double-stranded DNA inthe 5′ to 3′ direction.

In an aspect is provided a method of sequencing a templatepolynucleotide. In embodiments, the method includes: generating adouble-stranded amplification product including a first strandhybridized to a second strand, wherein (i) the double-strandedamplification product includes the template polynucleotide or complementthereof, and (ii) the first strand and second strand are both attachedto a solid support; generating a first invasion strand hybridized to thesecond strand by hybridizing one or more invasion primers (e.g., one ormore first invasion primers) to the second strand, and extending the oneor more invasion primers (e.g., extending the one or more first invasionprimers with a polymerase under strand-displacing conditions); andgenerating a first sequencing read by hybridizing one or more sequencingprimers (e.g., one or more first sequencing primers) to the firststrand, and extending the one or more first sequencing primers. Inembodiments, when hybridized to the second strand, the first invasionstrand blocks and/or prevents rehybridization of the complementary firststrand. In embodiments, the invasion primer is not covalently attachedto the solid support. In embodiments, the invasion strand, alternativelyreferred to herein as the third polynucleotide, is not covalentlyattached to the solid support.

In embodiments, the method includes: generating a double-strandedamplification product including a first strand hybridized to a secondstrand, wherein (i) the double-stranded amplification product includesthe template polynucleotide or complement thereof, and (ii) the firststrand and second strand are both attached to a solid support;generating a first invasion strand hybridized to the second strand byhybridizing an invasion primer to the second strand, and extending theinvasion primer, wherein the invasion primer is not covalently attachedto the solid support; and generating a first sequencing read byhybridizing one or more sequencing primers to the first strand, andextending the one or more first sequencing primers. In embodiments, theinvasion primer does not hybridize at the end of the strand, rather theinvasion primer hybridizes about 5 to about 50 nucleotides from the endof the strand. In embodiments, the invasion primer hybridizes about 10to about 30 nucleotides, about 12 to about 24, or about 15 to about 30from the end of the strand. In embodiments, the invasion primerhybridizes towards the 5′ end of the strand. In embodiments, theinvasion primer hybridizes towards the 3′ end of the strand. Inembodiments, the invasion primer does hybridize at the end of the strand(e.g., the invasion primer hybridizes to the last nucleotide on thestrand).

In embodiments, the method includes: generating a double-strandedamplification product including a first strand hybridized to a secondstrand, wherein (i) the double-stranded amplification product includesthe template polynucleotide or complement thereof, and (ii) the firststrand and second strand are both attached to a solid support;generating a first invasion strand hybridized to the second strand byhybridizing an invasion primer (e.g., a first invasion primer) to thesecond strand, and extending the first invasion primer (e.g., extendingthe first invasion primers with a polymerase under strand-displacingconditions); and generating a first sequencing read by hybridizing oneor more first sequencing primers to the first strand, and extending theone or more first sequencing primers. In embodiments, when hybridized tothe second strand, the first invasion strand blocks and/or preventsrehybridization of the complementary first strand. In embodiments, thefirst invasion primer is not covalently attached to the solid support.In embodiments, the invasion strand is not covalently attached to thesolid support.

In embodiments, each invasion primer of the one or more invasionsprimers is complementary to the same sequence (e.g., the same sequencein the first strand or the same sequence in the second strand). Inembodiments, each invasion primer of the one or more invasion primers isnot complementary to a different sequence (e.g., a different sequence inthe first strand or a different sequence in the second strand). Inembodiments, each invasion primer of the one or more invasion primers iscomplementary to a different sequence (e.g., a different sequence in thefirst strand or a different sequence in the second strand). Inembodiments, one or more invasions primers is complementary to the samesequence (e.g., the same sequence in the first strand or the samesequence in the second strand). In embodiments, one or more invasionprimers is not complementary to a different sequence (e.g., a differentsequence in the first strand or a different sequence in the secondstrand). In embodiments, one or more invasion primers is complementaryto a different sequence (e.g., a different sequence in the first strandor a different sequence in the second strand).

In embodiments, the first strand is covalently attached to the solidsupport via a first linker and the second strand is covalently attachedto the solid support via a second linker. The linker tethering thepolynucleotide strands may be any linker capable of localizing nucleicacids to arrays. The linkers may be the same, or the linkers may bedifferent. Solid-supported molecular arrays have been generatedpreviously in a variety of ways, for example, the attachment ofbiomolecules (e.g., proteins and nucleic acids) to a variety ofsubstrates (e.g., glass, plastics, or metals) underpins modernmicroarray and biosensor technologies employed for genotyping, geneexpression analysis and biological detection. Silica-based substratesare often employed as supports on which molecular arrays areconstructed, and functionalized silanes are commonly used to modifyglass to permit a click-chemistry enabled linker to tether thebiomolecule.

In embodiments, the method further includes generating a second invasionstrand hybridized to the first strand by hybridizing one or more secondinvasion primers to the first strand, and extending the one or moresecond invasion primers; and generating a second sequencing read byhybridizing one or more second sequencing primers to the second strand,and extending the one or more second sequencing primers. In embodiments,the second invasion strand is not covalently attached to the solidsupport. In embodiments, the method further includes removing the firstinvasion strand; generating a second invasion strand hybridized to thefirst strand by hybridizing one or more invasion primers to the firststrand, and extending the one or more second invasion primers; andgenerating a second sequencing read by hybridizing one or more secondsequencing primers to the second strand, and extending the one or moresecond sequencing primers. In embodiments, the method further includesgenerating a second invasion strand hybridized to the first strand byhybridizing a second invasion primer to the first strand, and extendingthe second invasion primer; and generating a second sequencing read byhybridizing one or more second sequencing primers to the second strand,and extending the one or more second sequencing primers. In embodiments,the second invasion strand is not covalently attached to the solidsupport. In embodiments, the method further includes removing the firstinvasion strand; generating a second invasion strand hybridized to thefirst strand by hybridizing a second invasion primer to the firststrand, and extending the second invasion primers; and generating asecond sequencing read by hybridizing one or more second sequencingprimers to the second strand, and extending the one or more secondsequencing primers. In embodiments, the method includes sequencing bothstrands (i.e., the first and the second strand) of the sampledouble-stranded amplification product. In embodiments, the methodincludes sequencing both strands (i.e., the first and the second strand)of the template polynucleotide.

In embodiments, the double-stranded amplification product includescommon sequences at their 5′ and 3′ ends. In this context the term“common” is interpreted as meaning common to all templates in thelibrary. For example, the double-stranded amplification product mayinclude a first adapter sequence at the 5′ end and a second adaptersequence at the 3′ end. Typically, the first adapter sequence and thesecond adapter sequence will consist of no more than 100, or no morethan 50, or no more than 40 consecutive nucleotides at the 5′ and 3′ends, respectively, of each strand of each template polynucleotide. Theprecise length of the two sequences may or may not be identical. Theprecise sequences of the common regions are generally not material tothe invention and may be selected by the user. The common sequences mustat least include primer-binding sequences (i.e., regions ofcomplementarity for a primer) which enable specific annealing of primerswhen the template polynucleotides are in used in a solid-phaseamplification reaction. The primer-binding sequences are thus determinedby the sequence of the primers to be ultimately used for solid-phaseamplification.

In embodiments, generating the invasion strand (i.e., generating thefirst invasion strand or the second invasion strand) includeshybridizing one or more primers to a common sequence in thedouble-stranded amplification product. In embodiments, generating theinvasion strand (i.e., generating the first invasion strand or thesecond invasion strand) includes hybridizing one primer to a commonsequence in the double-stranded amplification product. In embodiments,generating the invasion strand (i.e., generating the first invasionstrand or the second invasion strand) includes hybridizing a primer toat or near the 3′ end of the double-stranded amplification product. Inembodiments, generating the invasion strand (i.e., generating the firstinvasion strand or the second invasion strand) includes hybridizing aprimer to at or near the 3′ end of the double-stranded amplificationproduct, wherein the primer is not covalently attached to the solidsupport (e.g., the primer is in solution prior to hybridization). Inembodiments, the invasion primer does not hybridize at the terminus ofthe strand, rather the invasion primer hybridizes about 10, about 20,about 30, or about 50 nucleotides from the terminus of the strand. Inembodiments, the invasion primer hybridizes about 10 to about 30nucleotides from the terminus of the strand. In embodiments, theinvasion primer hybridizes to a common sequence (e.g., a sequencedescribed in U.S. Patent Publication 2016/0256846, which is incorporatedherein by reference, for example SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, or SEQ ID NO: 11 of U.S. Patent Publication 2016/0256846).

In embodiments, the method further includes removing the first strand bycleaving the first strand at a cleavable site, washing away the cleavedstrand, and generating a second sequencing read by hybridizing one ormore second sequencing primers to the second strand; and extending theone or more second sequencing primers. In embodiments, removing thefirst strand is optional. The one or more cleavable sites may include amodified nucleotide, ribonucleotide, or a sequence containing a modifiedor unmodified nucleotide that is specifically recognized by a cleavageagent. The cleavable site(s) may be deoxyuracil triphosphate (dUTP),deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), or other modifiednucleotide(s), such as those described, for example, in US 2012/0238738,which is incorporated herein by reference for all purposes. Inembodiments, the cleavable site includes a diol linker, disulfidelinker, photocleavable linker, abasic site, deoxyuracil triphosphate(dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylatednucleotide, ribonucleotide, or a sequence containing a modified orunmodified nucleotide that is specifically recognized by a cleavingagent. In embodiments, the cleavable site includes one or moreribonucleotides. In embodiments, the cleavable site includes 2 to 5ribonucleotides. In embodiments, the cleavable site includes oneribonucleotide. In embodiments, the cleavable sites can be cleaved at ornear a modified nucleotide or bond by enzymes or chemical reagents,collectively referred to here and in the claims as “cleaving agents.”Examples of cleaving agents include DNA repair enzymes, glycosylases,DNA cleaving endonucleases, or ribonucleases. For example, cleavage atdUTP may be achieved using uracil DNA glycosylase and endonuclease VIII(USER™, NEB, Ipswich, Mass.), as described in U.S. Pat. No. 7,435,572.In embodiments, when the modified nucleotide is a ribonucleotide, thecleavable site can be cleaved with an endoribonuclease. In embodiments,cleaving an extension product includes contacting the cleavable sitewith a cleaving agent, wherein the cleaving agent includes a reducingagent, sodium periodate, RNase, formamidopyrimidine DNA glycosylase(Fpg), endonuclease, restriction enzyme, or uracil DNA glycosylase(UDG). In embodiments, the cleaving agent is an endonuclease enzyme suchas nuclease P1, AP endonuclease, T7 endonuclease, T4 endonuclease IV,Bal 31 endonuclease, Endonuclease I (endo I), Micrococcal nuclease,Endonuclease II (endo VI, exo III), nuclease BAL-31 or mung beannuclease. In embodiments, the cleaving agent includes a restrictionendonuclease, including, for example a type IIS restrictionendonuclease. In embodiments, the cleaving agent is an exonuclease(e.g., RecBCD), restriction nuclease, endoribonuclease, exoribonuclease,or RNase (e.g., RNAse I, II, or III). In embodiments, the cleaving agentis a restriction enzyme. In embodiments, the cleaving agent includes aglycosylase and one or more suitable endonucleases. In embodiments,cleavage is performed under alkaline (e.g., pH greater than 8) bufferconditions at between 40° C. to 80° C. (e.g., 65° C.).

In embodiments, prior to generating a first invasion strand, the methodincludes removing immobilized primers that do not contain a first orsecond strand (i.e., unused primers). Methods of removing immobilizedprimers can include digestion using an enzyme with exonuclease activity.Removing unused primers may serve to increase the free volume and allowfor greater accessibility of the invasion primer. Removal of unusedprimers may also prevent opportunities for the newly released firststrand to rehybridize to an available surface primer, producing apriming site off the available surface primer, thereby facilitating the“reblocking” of the released first strand. In embodiments, prior togenerating a first invasion strand, the method includes contacting theimmobilized primers with an exonuclease enzyme.

In embodiments, prior to generating a first invasion strand, the methodincludes blocking the immobilized primers that do not include a first orsecond strand. In embodiments, the immobilized oligonucleotides includeblocking groups at their 3′ ends that prevent polymerase extension. Ablocking moiety prevents formation of a covalent bond between the 3′hydroxyl moiety of the nucleotide and the 5′ phosphate of anothernucleotide. In embodiments, prior to generating a first invasion strandthe method includes incubating the amplification products withdideoxynucleotide triphosphates (ddNTPs) to block the 3′-OH of theimmobilized oligonucleotides from future extension. In embodiments,prior to generating a first invasion strand, the method includesincorporating a dideoxynucleotide triphosphate (ddNTP) into animmobilized primer. In embodiments, prior to generating a first invasionstrand, the method includes contacting the immobilized primer with apolymerase. In embodiments, during generation of a first invasionstrand, the method includes contacting the immobilized primer with apolymerase buffer (e.g., incubating the solid support with a bufferedsolution including a polymerase).

In embodiments, the first strand is cleaved after generating the firstsequencing read but before generating the second sequencing read. Inembodiments, the first strand is not cleaved after generating the firstsequencing read. Cleaving one strand of the double-strandedamplification product may be referred to as linearization. Suitablemethods for linearization are known, and described in more detail inapplication number U.S. Patent Publication 2009/0118128, which isincorporated herein by reference in its entirety. For example, the firststrand may be cleaved by exposing the first strand to a mixturecontaining a glycosylase and one or more suitable endonucleases. Inembodiments, the first strand is attached to the surface in a way thatallows for selective removal. If the first template strand is removedfrom the surface, and the partially double-stranded amplificationproduct is denatured, for example by treatment with hydroxide orformamide, then the second strand remains immobilized as a linearizedsingle strand. If one of the surface immobilized primers includes acleavable site such that it can be cleaved from the surface, (e.g., diollinkage) the resulting partially double-stranded amplification productcan be made single-stranded using heat, or chemical denaturing agents,or a combination thereof providing conditions to give a single strandcontaining a primer hybridization site.

Any suitable enzymatic, chemical, or photochemical cleavage reaction maybe used to cleave the cleavable site. The cleavage reaction may resultin removal of a part or the whole of the strand being cleaved. Suitablecleavage means include, for example, restriction enzyme digestion, inwhich case the cleavable site is an appropriate restriction site for theenzyme which directs cleavage of one or both strands of a duplextemplate; RNase digestion or chemical cleavage of a bond between adeoxyribonucleotide and a ribonucleotide, in which case the cleavablesite may include one or more ribonucleotides; chemical reduction of adisulfide linkage with a reducing agent (e.g., THPP or TCEP), in whichcase the cleavable site should include an appropriate disulfide linkage;chemical cleavage of a diol linkage with periodate, in which case thecleavable site should include a diol linkage; generation of an abasicsite and subsequent hydrolysis, etc. In embodiments, the cleavable siteis included in the surface immobilized primer (e.g., within thepolynucleotide sequence of the primer). In embodiments, one strand ofthe double-stranded amplification product (or the surface immobilizedprimer) may include a diol linkage which permits cleavage by treatmentwith periodate (e.g., sodium periodate). It will be appreciated thatmore than one diol can be included at the cleavable site. One or morediol units may be incorporated into a polynucleotide using standardmethods for automated chemical DNA synthesis. Polynucleotide primersincluding one or more diol linkers can be conveniently prepared bychemical synthesis. The diol linker is cleaved by treatment with anysubstance which promotes cleavage of the diol (e.g., a diol-cleavingagent). In embodiments, the diol-cleaving agent is periodate, e.g.,aqueous sodium periodate (NaIO₄). Following treatment with thediol-cleaving agent (e.g., periodate) to cleave the diol, the cleavedproduct may be treated with a “capping agent” in order to neutralizereactive species generated in the cleavage reaction. Suitable cappingagents for this purpose include amines, e.g., ethanolamine orpropanolamine.

In embodiments, the cleavable site is not in the immobilized primersequence (e.g., within the polynucleotide sequence of the primer). Inembodiments, the cleavable site is included in the linking moietyresponsible for tethering the primer to the substrate. In embodiments,the cleavable site is a cleavable linker (e.g., a disulfide containinglinker that cleaves when exposed to a reducing agent). In embodiments,the cleavable site is a diol linker.

In embodiments, the first strand includes at least one cleavable site.In embodiments, the first linker includes at least one cleavable site.In embodiments, the cleavable site includes deoxyuracil triphosphate(dUTP). The enzyme uracil DNA glycosylase (UDG) may then be used toremove dUTP, generating an abasic site on one strand. The polynucleotidestrand including the abasic site may then be cleaved at the abasic siteby treatment with endonuclease (e.g EndoIV endonuclease, AP lyase, FPGglycosylase/AP lyase, EndoVIII glycosylase/AP lyase), heat or alkali. Inembodiments, the USER™ reagent available from New England Biolabs (NEBcatalog #M5508) is used for the creation of a single nucleotide gap at auracil base in a duplex strand.

In embodiments, the cleavable site includes a diol linker, disulfidelinker, photocleavable linker, abasic site, deoxyuracil triphosphate(dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylatednucleotide, ribonucleotide, or a sequence containing a modified orunmodified nucleotide that is specifically recognized by a cleavingagent.

In embodiments, the cleavable site includes one or more ribonucleotides.In embodiments, the cleavable site includes 2 to 5 ribonucleotides. Inembodiments, the cleavable site includes one ribonucleotide. Inembodiments, the cleavable site includes more than one ribonucleotide.In embodiments, the cleavable site includes deoxyuracil triphosphate(dUTP) or deoxy-8-oxo-guanine triphosphate (d-8-oxoG). In embodiments,the cleavable site includes two or more deoxyuracil triphosphate (dUTP).In embodiments, the cleavable site includes 2 to 15 dUTPs.

In embodiments, cleaving includes enzymatically cleaving the firststrand at the at least one cleavable site (e.g., enzymatically cleavingwith an endonuclease). In embodiments, the first strand includes a diollinker, disulfide linker, photocleavable linker, abasic site,deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate(d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequencecontaining a modified or unmodified nucleotide that is specificallyrecognized by a cleaving agent.

In embodiments, cleaving the first strand includes contacting thecleavable site with a cleaving agent, wherein the cleaving agentincludes a reducing agent, sodium periodate, RNase, FormamidopyrimidineDNA Glycosylase (Fpg), endonuclease, restriction enzyme, or uracil DNAglycosylase (UDG). In embodiments, the cleaving agent is an endonucleaseenzyme such as nuclease P1, AP endonuclease, T7 endonuclease, T4endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I),Micrococcal nuclease, Endonuclease II (endo VI, exo III), nucleaseBAL-31 or mung bean nuclease. In embodiments, the cleaving agentincludes a restriction endonuclease, including, for example a type IISrestriction endonuclease. In embodiments, the cleaving agent is anexonuclease (e.g., RecBCD), restriction nuclease, endoribonuclease,exoribonuclease, or RNase (e.g., RNAse I, II, or III). In embodiments,the cleaving agent is a restriction enzyme. In embodiments, the cleavingagent includes a glycosylase and one or more suitable endonucleases. Inembodiments, cleavage is performed under alkaline (e.g., pH greater than8) buffer conditions at between 40° C. to 80° C. (e.g., 65° C.).

In embodiments, cleaving includes chemically cleaving the first strandat the at least one cleavable site. In embodiments, the first linkerincludes a diol linker, disulfide linker, photocleavable linker, abasicsite, deoxyuracil triphosphate (dUTP), deoxy-8-Oxo-guanine triphosphate(d-8-oxoG), methylated nucleotide, ribonucleotide, or a sequencecontaining a modified or unmodified nucleotide that is specificallyrecognized by a cleaving agent.

In embodiments, the invasion primer is not covalently attached to thesolid support. In embodiments, the invasion primer includes syntheticnucleotides. In embodiments, the invasion primer includes locked nucleicacids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalatingnucleic acids (TINAs), bridged nucleic acids (BNAs), 2′-O-methyl RNA:DNAchimeric nucleic acids, minor groove binder (MGB) nucleic acids,morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptidenucleic acids (PNAs), or combinations thereof. In embodiments, theinvasion primer includes locked nucleic acids (LNAs), Bis-locked nucleicacids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridgednucleic acids (BNAs), peptide nucleic acids (PNAs), or combinationsthereof. In embodiments, the invasion primer includes locked nucleicacids (LNAs). In embodiments, the invasion primer includes Bis-lockednucleic acids (bisLNAs). In embodiments, the invasion primer includestwisted intercalating nucleic acids (TINAs). In embodiments, theinvasion primer includes bridged nucleic acids (BNAs). In embodiments,the invasion primer includes 2′-O-methyl RNA:DNA chimeric nucleic acids.In embodiments, the invasion primer includes minor groove binder (MGB)nucleic acids. In embodiments, the invasion primer includes morpholinonucleic acids. In embodiments, the invasion primer includes C5-modifiedpyrimidine nucleic acids. In embodiments, the invasion primer includespeptide nucleic acids (PNAs). In embodiments, the invasion primerincludes locked nucleic acids (LNAs), Bis-locked nucleic acids(bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleicacids (BNAs), peptide nucleic acids (PNAs), or combinations thereof.

In embodiments, the invasion primer includes locked nucleic acids(LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalatingnucleic acids (TINAs), bridged nucleic acids (BNAs), 2′-O-methyl RNA:DNAchimeric nucleic acids, minor groove binder (MGB) nucleic acids,morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptidenucleic acids (PNAs), phosphorothioate nucleic acids, or combinationsthereof. In embodiments, the invasion primer includes phosphorothioatenucleic acids. In embodiments, the invasion primer includes one or morelocked nucleic acids (LNAs), 2-amino-deoxyadenosine (2-amino-dA),trimethoxystilbene-functionalized oligonucleotides (TFOs),Pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids(PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids. Inembodiments, the invasion primer includes one or more locked nucleicacids (LNAs). In embodiments, the invasion primer includes one or more2-amino-deoxyadenosine (2-amino-dA). In embodiments, the invasion primerincludes one or more trimethoxystilbene-functionalized oligonucleotides(TFOs). In embodiments, the invasion primer includes one or morePyrene-functionalized oligonucleotides (PFOs). In embodiments, theinvasion primer includes one or more peptide nucleic acids (PNAs). Inembodiments, the invasion primer includes one or moreaminoethyl-phenoxazine-dC (AP-dC) nucleic acids. In embodiments, theinvasion primer includes 10 to 15 locked nucleic acids (LNAs). Inembodiments, the invasion primer includes a sequence described herein,for example within Table 1. In embodiments, the invasion primer includesone or more phosphorothioates at the 5′ end. In embodiments, theinvasion primer includes one or more LNAs at the 5′ end. In embodiments,the invasion primer includes two or more consecutive LNAs at the 3′ end.In embodiments, the invasion primer includes two to four consecutiveLNAs at the 3′ end. In embodiments, the invasion primer includes two ormore consecutive LNAs at the 5′ end. In embodiments, the invasion primerincludes two to four consecutive LNAs at the 5′ end.

In embodiments, the invasion primer includes one or more locked nucleicacids (LNAs) at the 3′ end of the invasion primer sequence. Inembodiments, the invasion primer includes 2, 3, 4, 5, or more lockednucleic acids (LNAs) at the 3′ end of the invasion primer sequence. Inembodiments, the invasion primer includes a plurality of locked nucleicacids (LNAs) at the 3′ end of the invasion primer sequence. Inembodiments, the invasion primer includes one locked nucleic acid (LNA)at the 3′ end of the invasion primer sequence. In embodiments, theinvasion primer includes 2 locked nucleic acids (LNAs) at the 3′ end ofthe invasion primer sequence. In embodiments, the invasion primerincludes 3 locked nucleic acids (LNAs) at the 3′ end of the invasionprimer sequence. In embodiments, the invasion primer includes 4 lockednucleic acids (LNAs) at the 3′ end of the invasion primer sequence. Inembodiments, the invasion primer includes 5 locked nucleic acids (LNAs)at the 3′ end of the invasion primer sequence.

In embodiments, the invasion primer includes from 5′ to 3′ a pluralityof synthetic nucleotides (e.g., LNAs) followed by a plurality (e.g., 2to 5) canonical or native nucleotides (e.g., dNTPs). In embodiments, theinvasion primer comprises one or more (e.g., 2 to 5) deoxyuracilnucleobases (dU). In embodiments, the one or more dU nucleobases are ator near the 3′ end of the invasion primer (e.g., within 5 nucleotides ofthe 3′ end). In embodiments, the invasion primer includes from 5′ to 3′a plurality (e.g., 2 to 5) of phosphorothioate nucleic acids, followedby a plurality of synthetic nucleotides (e.g., LNAs), and subsequentlyfollowed by a plurality (e.g., 2 to 5) of canonical bases. In someembodiments, the invasion primer includes a plurality of canonicalbases, wherein the canonical bases terminate (i.e., at the 3′ end) witha deoxyuracil nucleobase (dU).

In embodiments, the invasion primer includes the sequence provided inTable 1. In embodiments, the 5′ end of the sequences provided in Table 1include one or more phosphorothioate nucleic acids.

TABLE 1Invasion primer sequences, from 5′-3′, wherein the nucleotide contained inbrackets indicates an LNA nucleotide. SEQ IDNucleotide Sequence 5′ to 3′ numberT[T]T[T]T[C]T[C]CA[G]CG[A]GATG[A]CCCT[C]A[C]CAAC[C][A][C] SEQ ID NO: 1TTT[T]T[C]T[C]CA[G]CG[A]GATG[A]CCCT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 2TTTTT[C]T[C]CA[G]CG[A]GATG[A]C[C]CT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 3TTTTTCT[C]CA[G]CG[A]G[A]TG[A]C[C]CT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 4T[T]T[T]T[C]T[C]CA[G]CG[A]GATG[A]CCCT[C]A[C]C[A]AC[C][A][C] SEQ ID NO: 5T[T]T[T]T[C]T[C]CA[G]CG[A]GATG[A]C[C]CT[C]A[C]C[A]AC[C][A][C]SEQ ID NO: 6T[T]T[T]T[C]T[C]CA[G]CG[A]G[A]TG[A]C[C]CT[C]A[C]C[A]AC[C][A][C]SEQ ID NO: 7 T[T]T[T]TCUCC[A]G[C][G][A]GAUGA[C]CCUCA[C][C][A]A[C][C]ACUSEQ ID NO: 8 A[C]AC[T]CT[T]TC[C]CT[A]C[A]CGA[C]GC[T]CTT[C]CGATCTSEQ ID NO: 9 G[T]G[A]C[T]G[G]AG[T]TC[A]GACG[T]GTGC[T]C[T]TCCG[A]TCTSEQ ID NO: 10 G[T]G[A]C[T]G[G]AG[T]TC[A]GACG[T]GTGC[T]C[T]TCCG[A][T][C]SEQ ID NO: 11 CA[G]CG[A]GATG[A]CCCT[C]A[C]CAAC[C][A][C] SEQ ID NO: 12CA[G]CG[A]GATG[A]CCCT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 13CA[G]CG[A]GATG[A]C[C]CT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 14CA[G]CG[A]G[A]TG[A]C[C]CT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 15CA[G]CG[A]GATG[A]CCCT[C]A[C]C[A]AC[C][A][C] SEQ ID NO: 16CA[G]CG[A]GATG[A]C[C]CT[C]A[C]C[A]AC[C][A][C] SEQ ID NO: 17CA[G]CG[A]G[A]TG[A]C[C]CT[C]A[C]C[A]AC[C][A][C] SEQ ID NO: 18C[A]G[C][G][A]GAUGA[C]CCUCA[C][C][A]A[C][C]ACU SEQ ID NO: 19[T]TC[C]CT[A]C[A]CGA[C]GC[T]CTT[C]CGATCT SEQ ID NO: 20AG[T]TC[A]GACG[T]GTGC[T]C[T]TCCG[A]TCT SEQ ID NO: 21AG[T]TC[A]GACG[T]GTGC[T]C[T]TCCG[A][T][C] SEQ ID NO: 22T[T]T[T]T[C]T[C]CA[G]CG[A]GATG SEQ ID NO: 23TTT[T]T[C]T[C]CA[G]CG[A]GATG SEQ ID NO: 24 TTTTT[C]T[C]CA[G]CG[A]GATGSEQ ID NO: 25 TTTTTCT[C]CA[G]CG[A]G[A]TG SEQ ID NO: 26T[T]T[T]T[C]T[C]CA[G]CG[A]GATG SEQ ID NO: 27T[T]T[T]T[C]T[C]CA[G]CG[A]GATG SEQ ID NO: 28T[T]T[T]T[C]T[C]CA[G]CG[A]G[A]TG SEQ ID NO: 29T[T]T[T]TCUCC[A]G[C][G][A]GAUG SEQ ID NO: 30A[C]AC [T]CT[T]TC[C]CT[A]C [A] SEQ ID NO: 31 G[T]G[A]C[T]G[G]AG[T]TC[A]SEQ ID NO: 32 G[T]G[A]C[T]G[G]AG[T]TC[A] SEQ ID NO: 33AATGATACGGCGACCACCG SEQ ID NO: 34 CAAGCAGAAGACGGCATACGAGAT SEQ ID NO: 35CGGTGGTCGCCGTATCATT SEQ ID NO: 36 ATCTCGTATGCCGTCTTCTGCTTG SEQ ID NO: 37[T]T[T]T[C]T[C]CA[G]CG[A]GATG[A]CCCT[C]A[C]CAAC[C][A][C] SEQ ID NO: 38[T]T[C]T[C]CA[G]CG[A]GATG[A]CCCT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 39[C]T[C]CA[G]CG[A]GATG[A]C[C]CT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 40[C]CA[G]CG[A]G[A]TG[A]C[C]CT[C]A[C]CA[A]C[C][A][C] SEQ ID NO: 41[T]T[T]T[C]T[C]CA[G]CG[A]GATG[A]CCCT[C]A[C]C[A]AC[C][A][C] SEQ ID NO: 42[T]T[T]T[C]T[C]CA[G]CG[A]GATG[A]C[C]CT[C]A[C]C[A]AC[C][A][C]SEQ ID NO: 43[T]T[T]T[C]T[C]CA[G]CG[A]G[A]TG[A]C[C]CT[C]A[C]C[A]AC[C][A][C]SEQ ID NO: 44 AATGATAC[G]GCG[A]CCACC[G] SEQ ID NO: 45AA[T]GA[T]ACGGC[G]ACCAC[C][G] SEQ ID NO: 46[A]AU[G]AUA[C]GG[C]GACC[A]C[C]G SEQ ID NO: 47 CAAGCAGAAGACGGCATACGAGA[T]SEQ ID NO: 48 CAAG[C]AGA[A]G[A]CGGCATACG[A]GAT SEQ ID NO: 49TCA[A]GCAGA[A]GACGGCA[T][A][C]GA[G]A[T] SEQ ID NO: 50T[T]CA[A][G]CAGAAGA[C]GGCAUACGA[G][A]U SEQ ID NO: 51CGGTGGTCGCCGTATCA[T][T] SEQ ID NO: 52 C[G]GT[G]GUCGCC[G]TAUCAUUSEQ ID NO: 53 TT[T]CGGT[G]GT[C]GCCGTATCA[T][T] SEQ ID NO: 54[C]G[G]TGGUCGCCG[T]ATCA[T]T SEQ ID NO: 55A[T]CT[C]GT[A]TGCC[G]TCT[T]CTGCTT[G] SEQ ID NO: 56ATC[T]CGTATGCCGTCTTCTGCT[T][G] SEQ ID NO: 57ATCTC[G]TA[T]GC[C]GT[C]TTC[T]GC[T]T[G] SEQ ID NO: 58ATC[T]CGUAUGC[C]GTCTUTCUGCUU[G] SEQ ID NO: 59

In embodiments, the invasion primer includes one or more morpholinonucleic acids. Morpholino nucleic acids are synthetic nucleotides thathave standard nucleic acid bases (e.g., adenine, guanine, cytosine, andthymine) wherein those bases are bound to methylenemorpholine ringslinked through phosphorodiamidate groups instead of phosphates.Morpholino nucleic acids may be referred to as phosphorodiamidatemorpholino oligomers (PMOs).

In embodiments, the invasion primer includes locked nucleic acids(LNAs). In embodiments, the invasion primer includes LNAs dispersedthroughout the primer, wherein about 2 to 5 nucleotides on the 3′ endare canonical dNTPs. In embodiments, the entire composition of theinvasion primer includes less than 50%, less than 40%, or less than 30%of LNAs.

In embodiments, the invasion primer includes peptide nucleic acids(PNAs). A PNA is a synthetic nucleic acid analogue wherein thenucleobases are arrayed along a neutral N-(2-aminoethyl)-glycinebackbone in place of the negatively charged phosphate backbone ofcanonical DNA. The unique pseudopeptide backbone is considered to beresponsible for dramatically altering the interactions of nucleic acidsand proteins with PNA. For example resulting in increasedthermostability of PNA hybridization with DNA. It is known that PNAhybridization demonstrates a negative salt dependence wherein lowerionic strength results in increased duplex stability (see, for example,De Costa N. T. S. Heemstra J. M. PLoS One. 2013; 8:e58670. Inembodiments, the invasion primer includes one or more PNAs and annealsto the dsDNA (e.g., the second strand) in a buffer containing less than200 nM NaCl, less than about 100 nM NaCl, or less than about 50 nM NaCl.

In embodiments, the invasion primer includes a plurality of LNAsinterspersed throughout the polynucleotide. In embodiments, the invasionprimer includes a plurality of consecutive LNAs (e.g., 2 to 5 LNAs, 5 to7 LNAs, or 7 to 10 LNAs) throughout the polynucleotide. In embodiments,the entire composition of the invasion primer includes less than 70%,less than 60%, less than 50%, less than 40%, less than 30%, less than20%, less than 10%, or less than 5% of LNAs. In embodiments, the entirecomposition of the invasion primer includes up to about 70%, up to about60%, up to about 50%, up to about 40%, up to about 30%, up to about 20%,up to about 10%, or up to about 5% of LNAs. In embodiments, the entirecomposition of the invasion primer includes more than 60%, more than50%, more than 40%, more than 30%, more than 20%, more than 10%, or morethan 5% of LNAs. In embodiments, the entire composition of the invasionprimer includes about 5% to about 10%, about 10% to about 20%, about 20%to about 30%, about 30% to about 40%, about 40% to about 50%, about 50%to about 60%, or about 60% to about 70% of LNAs. In embodiments, theentire composition of the invasion primer includes about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 65%, or about 70% of LNAs.In embodiments, the entire composition of the invasion primer includesabout 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,or about 95% of canonical dNTPs. In embodiments, the entire compositionof the invasion primer includes less than 95%, less than 90%, less than80%, less than 70%, less than 60%, less than 50%, less than 40%, or lessthan 30% of canonical dNTPs. In embodiments, the entire composition ofthe invasion primer includes up to about 95%, up to about 90%, up toabout 80%, up to about 70%, up to about 60%, up to about 50%, up toabout 40%, or up to about 30% of canonical dNTPs. In embodiments, theentire composition of the invasion primer includes more than 90%, morethan 80%, more than 70%, more than 60%, more than 50%, more than 40%, ormore than 30% of canonical dNTPs.

In embodiments, the invasion primer includes about 70% of LNAs and about30% of canonical dNTPs. In embodiments, the invasion primer includesabout 65% of LNAs and about 35% of canonical dNTPs. In embodiments, theinvasion primer includes about 60% of LNAs and about 40% of canonicaldNTPs. In embodiments, the invasion primer includes about 55% of LNAsand about 45% of canonical dNTPs. In embodiments, the invasion primerincludes about 50% of LNAs and about 50% of canonical dNTPs. Inembodiments, the invasion primer includes about 45% of LNAs and about55% of canonical dNTPs. In embodiments, the invasion primer includesabout 40% of LNAs and about 60% of canonical dNTPs. In embodiments, theinvasion primer includes about 35% of LNAs and about 65% of canonicaldNTPs. In embodiments, the invasion primer includes about 30% of LNAsand about 70% of canonical dNTPs. In embodiments, the invasion primerincludes about 25% of LNAs and about 75% of canonical dNTPs. Inembodiments, the invasion primer includes about 20% of LNAs and about80% of canonical dNTPs. In embodiments, the invasion primer includesabout 15% of LNAs and about 85% of canonical dNTPs. In embodiments, theinvasion primer includes about 10% of LNAs and about 90% of canonicaldNTPs. In embodiments, the invasion primer includes about 5% of LNAs andabout 95% of canonical dNTPs.

In embodiments, the invasion primer includes one or more dT nucleobasesthat are replaced with dU nucleobases. In embodiments, the invasionprimer includes a plurality of dT nucleobases that are replaced with dUnucleobases. In embodiments, the invasion primer includes all dTnucleobases replaced with dU nucleobases. In embodiments, the one ormore dU nucleobases partition the invasion primer into two or moreregions of consecutive nucleotides (e.g., a first plurality ofconsecutive nucleotides and a second plurality of consecutivenucleotides are separated by the one or more dU nucleobases). Inembodiments each of the two or more regions of consecutive nucleotidesare each about 3 to about 10 nucleotides in length, or about 3 to about15 nucleotides in length. In embodiments each of the two or more regionsof consecutive nucleotides are each about 3 to about 10 nucleotides inlength. In embodiments each of the two or more regions of consecutivenucleotides are each about 3 to about 15 nucleotides in length. Inembodiments each of the two or more regions of consecutive nucleotidesare each at least about 3, 5, 7, 10, 13, or 15 nucleotides in length. Inembodiments, each of the two or more regions of consecutive nucleotidesis greater than about 15 nucleotides in length. In embodiments, thecalculated or predicted melting temperature (Tm) of each of the two ormore regions of consecutive nucleotides is about 50° C. to about 75° C.In embodiments, the calculated or predicted melting temperature (Tm) ofeach of the two or more regions of consecutive nucleotides is about 60°C. to about 75° C. In embodiments, the calculated or predicted meltingtemperature (Tm) of each of the two or more regions of consecutivenucleotides is about 50° C. to about 65° C. In embodiments, thecalculated or predicted melting temperature (Tm) of each of the two ormore regions of consecutive nucleotides is less than about 75° C. Inembodiments, the calculated or predicted melting temperature (Tm) ofeach of the two or more regions of consecutive nucleotides is less thanabout 65° C. In embodiments, the calculated or predicted meltingtemperature (Tm) of each of the two or more regions of consecutivenucleotides is less than about 60° C. In embodiments, the dU and the LNAnucleotides are not adjacent to each other. In embodiments, the dU andthe LNA nucleotides are separated by one or more native nucleotides.

In embodiments, the invasion primer is about 10 to 100 nucleotides inlength. In embodiments, the invasion primer is about 15 to about 75nucleotides in length. In embodiments, the invasion primer is about 25to about 75 nucleotides in length. In embodiments, the invasion primeris about 15 to about 50 nucleotides in length. In embodiments, theinvasion primer is about 10 to about 20 nucleotides in length. Inembodiments, the invasion primer is about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or about 20 nucleotides in length. In embodiments, theinvasion primer is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, orabout 30 nucleotides in length. In embodiments, the invasion primer isabout 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 nucleotides inlength. In embodiments, the invasion primer is about 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or about 50 nucleotides in length. In embodiments,the invasion primer is greater than 30 nucleotides in length. Inembodiments, the invasion primer is greater than 40 nucleotides inlength. In embodiments, the invasion primer is greater than 50nucleotides in length. In embodiments, the invasion primer is no lessthan 20 nucleotides. In embodiments, the invasion primer is about 15 toabout 35 nucleotides in length. In embodiments, the invasion primer isabout 25 to about 35 nucleotides, wherein 12 to 18 nucleotides are LNAnucleotides. In embodiments, the invasion primer is about 25 to about 35nucleotides, wherein 14 to 16 nucleotides are LNA nucleotides. Inembodiments, the invasion primer is about 30 to about 35 nucleotides,wherein 14 to 16 nucleotides are LNA nucleotides. In embodiments, theinvasion primer is 30, 31, 32, or 33 nucleotides, wherein 14 to 16nucleotides are LNA nucleotides.

In embodiments, the calculated or predicted melting temperature (Tm) ofthe invasion primer is about 70° C. to about 95° C. In embodiments, thecalculated or predicted melting temperature (Tm) of the invasion primeris about 80° C. to about 95° C. In embodiments, the calculated orpredicted melting temperature (Tm) of the invasion primer is about 85°C. to about 95° C. In embodiments, the calculated or predicted meltingtemperature (Tm) of the invasion primer is about 85° C. to about 90° C.

In embodiments, the method includes generating a first invasion strandby hybridizing a first invasion primer (e.g., an invasion primer thatincludes one or more PNAs or LNAs) to the first strand. In embodiments,the invasion primer does not hybridize at the end of the strand, ratherthe invasion primer hybridizes about 5 to about 50 nucleotides from theend of the strand. In embodiments, the invasion primer hybridizes about10 to about 30 nucleotides, about 12 to about 24, or about 15 to about30 from the end of the strand. The first invasion primer creates a“bubble” in the duplex (e.g., as depicted in FIGS. 4A-4B). A secondinvasion primer anneals to the second strand (e.g., within the bubbleformed by annealing the first invasion primer) and is extended therebygenerating a first invasion strand hybridized to the second strand. Thefirst invasion primer may remain during the first sequencing read, ormay be removed prior to starting the first sequencing read. Inembodiments, the first invasion primer and the second invasion primerare not covalently attached to the solid support.

In embodiments, generating the invasion strand includes a plurality ofinvasion primer extension cycles, wherein each invasion primer extensioncycle includes incorporating one or more nucleotides into the invasionprimer. In embodiments, generating the invasion strand includesextending the invasion primer by incorporating one or more nucleotides(e.g., dNTPs) using Bst large fragment (Bst LF) polymerase, Bst2.0polymerase, Bsu polymerase, SD polymerase, Vent exo-polymerase, Phi29polymerase, or a mutant thereof. In embodiments, the polymerase extendsby incorporating a nucleotide to the 3′ end of the invasion primer. Inembodiments, the polymerase extends by incorporating a nucleotide to the3′ end of an LNA nucleotide of the invasion primer.

In embodiments, generating the invasion strand includes a plurality ofinvasion-primer extension cycles by incorporating universal nucleobases(e.g., 5-nitroindole and/or inosine nucleobases) into the invasionprimer. The blocking strand does not need to be a faithfulrepresentation (i.e., an exact copy) of the strand to which the invasionprimer is hybridized. In the interest of speed, in embodiments, one ormore inosine nucleotides or “universal” nucleotides may be incorporatedinto the primer to generate a blocking strand. The term “universalnucleotide,” as used herein, refers to a nucleotide analog that iscapable of forming a base pair to two or more (e.g., any of the four)natural nucleotide bases (e.g., cytosine (C), guanine (G), adenine (A),or thymine (T)). Thus, any other base may be paired with a universalbase analog in a double-stranded polynucleotide. Universal nucleotidesmay be divided into hydrogen bonding bases and pi-stacking bases.Hydrogen bonding bases form hydrogen bonds with any of the naturalnucleobases. The hydrogen bonds formed by hydrogen bonding bases areweaker than the hydrogen bonds between natural nucleobases. Pi-stackingnucleobases are non-hydrogen bonding, hydrophobic, aromatic bases thatstabilize duplex polynucleotides by stacking interactions. Examples ofhydrogen bonding bases include, but are not limited to, hypoxanthine(inosine), 7-deazahypoxanthine, 2-azahypoxanthine, 2-hydroxypurine,purine, and 4-Amino-1H-pyrazolo [3,4-d]pyrimidine. IExamples ofpi-stacking bases include, but are not limited to, nitroimidazole,indole, benzimidazole, 5-fluoroindole, 5-nitroindole,N-indol-5-yl-formamide, isoquinoline, and methylisoquinoline. Examplesof universal bases are discussed in Berger et al., Universal Bases forHybridization, Replication and Chain Termination, Nucleic Acids Research2000, August 1, 28(15) pp. 2911-2914; David Loakes, The Applications ofUniversal DNA Base Analogs, 29(12) Nucleic Acids Research 2437 (2001);and Feng Liang et al., Universal base analogs and their applications inDNA sequencing technology, 3 RSC Advances 14910-14928 (2013). Inembodiments, the invasion strand includes at least a subset ofnucleotides that are not universal nucleotides. In embodiments, at least1% to 10% of the nucleotides in the invasion strand are universalnucleotides. In embodiments, at least 50% of the nucleotides in theinvasion strand are not universal nucleotides.

In embodiments, the blocking strand includes universal nucleobases. Inembodiments, the invasion strand is generated using an error-pronepolymerase, for example Taq, a Y-family member Dpo4, or others known inthe art (e.g., Rattray A J and Strathern J N. Annu Rev Genet. 2003;37:31-66). In embodiments, the blocking strand is not a copy of thestrand the invasion primer is hybridized to. In embodiments, theblocking strand does not replicate the exact sequence of the strand towhich the invasion primer is hybridized.

In embodiments, generating the invasion strand includes a firstplurality of invasion-primer extension cycles followed by a secondplurality of invasion-primer extension cycles, wherein the reactionconditions for the first plurality of invasion-primer extension cyclesare different than the second plurality of invasion-primer extensioncycles. In embodiments, generating the invasion strand includesalternating between a first plurality of invasion-primer extensioncycles and a second plurality of invasion-primer extension cycles,wherein the reaction conditions for the first plurality ofinvasion-primer extension cycles are different than the second pluralityof invasion-primer extension cycles. In embodiments, the reactionconditions for the first plurality of invasion-primer extension cyclesinclude higher stringency hybridization conditions relative to thesecond plurality of invasion-primer extension cycles.

In embodiments, the reaction conditions for the first plurality ofinvasion-primer extension cycles include incubation in a firstdenaturant. In embodiments, the first denaturant includes additives suchas ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethylsulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine,or tetramethylammonium chloride (TMAC). In embodiments, the firstdenaturant is a buffered solution including about 0% to about 50%dimethyl sulfoxide (DMSO); about 0% to about 50% ethylene glycol; about0% to about 20% formamide; or about 0 to about 3M betaine, or a mixturethereof. In embodiments, the reaction conditions for the first pluralityof invasion-primer extension cycles include incubation in a firstdenaturant, wherein the first denaturant is a buffered solutionincluding about 15% to about 50% dimethyl sulfoxide (DMSO); about 15% toabout 50% ethylene glycol; about 10% to about 20% formamide; or about 0to about 3M betaine, or a mixture thereof. In embodiments, thetemperature is between 50° C. and about 75° C., inclusive of theendpoints (i.e., the temperature may be 50° C., 52° C., or 75° C.,etc.). In embodiments, the temperature is about 50° C. to about 75° C.In embodiments, the temperature is about 55° C. to about 70° C. Inembodiments, the temperature is about 60° C. to about 70° C. Inembodiments, the temperature is about 55° C. to about 68° C. Inembodiments, the buffered solution includes 5×SSC.

In embodiments, the reaction conditions for the second plurality ofinvasion-primer extension cycles include incubation in a seconddenaturant. In embodiments, the second denaturant includes additivessuch as ethylene glycol, polyethylene glycol, 1,2-propanediol, dimethylsulfoxide (DMSO), glycerol, formamide, 7-deaza-dGTP, acetamide, betaine,or tetramethylammonium chloride (TMAC), wherein the concentrations ofthe additives in the second denaturant differ than the concentrations ofthe additives in the first denaturant. In embodiments, the seconddenaturant is a buffered solution including about 0 to about 50%dimethyl sulfoxide (DMSO); about 0 to about 50% ethylene glycol; about 0to about 20% formamide; or about 0 to about 3M betaine, or a mixturethereof. In embodiments, the reaction conditions for the secondplurality of invasion-primer extension cycles include incubation in asecond denaturant, wherein the second denaturant is a buffered solutionincluding about 0% to about 15% dimethyl sulfoxide (DMSO); about 0 toabout 15% ethylene glycol; about 0 to about 10% formamide; or about 0 toabout 3M betaine, or a mixture thereof. In embodiments, the temperatureis between 50° C. and about 75° C., inclusive of the endpoints (i.e.,the temperature may be 50° C., 52° C., or 75° C., etc.). In embodiments,the temperature is about 50° C. to about 75° C. In embodiments, thetemperature is about 55° C. to about 70° C. In embodiments, thetemperature is about 60° C. to about 70° C. In embodiments, thetemperature is about 55° C. to about 68° C. In embodiments, the bufferedsolution includes 5×SSC.

In embodiments, the first denaturant is a buffered solution includingdimethyl sulfoxide (DMSO); and the second denaturant is a bufferedsolution including dimethyl sulfoxide (DMSO) and betaine. Inembodiments, the first denaturant is a buffered solution including about25 to about 35% DMSO; and the second denaturant is a buffered solutionincluding about 0 to about 10% DMSO and about 1M to about 4M betaine. Inembodiments, the first denaturant is a buffered solution including about30% DMSO; and the second denaturant is a buffered solution includingabout 5% DMSO, about 2.5M betaine.

In embodiments, the reaction conditions for the second plurality ofinvasion-primer extension cycles further includes incubation with a SSBprotein.

In embodiments, generating the invasion strand (e.g., the first invasionstrand and/or the second invasion strand) comprises contacting thepolynucleotide with one or more invasion-reaction mixtures. Inembodiments, generating the invasion strand includes contacting thedouble-stranded amplification product with one or more invasion-reactionmixtures; each of the invasion-reaction mixture including a plurality ofinvasion primers, a plurality of deoxyribonucleotide triphosphate(dNTPs), and a polymerase. In embodiments, generating the invasionstrand includes contacting the double-stranded amplification productwith a first invasion-reaction mixture followed by contacting thedouble-stranded amplification product with a second invasion-reactionmixture; the first invasion-reaction mixture including a plurality ofinvasion primers and no polymerase; and the second invasion-reactionmixture includes a plurality of deoxyribonucleotide triphosphate (dNTPs)and a polymerase. In embodiments, the polymerase is a strand-displacingpolymerase. In embodiments, the strand-displacing polymerase is Bstlarge fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0polymerase, Bsu polymerase, SD polymerase, Vent exo-polymerase, Phi29polymerase, or a mutant thereof.

In embodiments, each of the plurality of invasion-reaction mixturesinclude a plurality of invasion primers, a plurality ofdeoxyribonucleotide triphosphate (dNTPs), a polymerase, or a combinationthereof. In embodiments, each of the plurality of invasion-reactionmixtures include a denaturant, single-stranded DNA binding protein(SSB), or both a denaturant and single-stranded DNA binding protein(SSB). In embodiments, each invasion-reaction mixture further includes adenaturant, single-stranded DNA binding protein (SSB), or a combinationthereof. In embodiments, each invasion-reaction mixture includes adifferent amount of a denaturant, single-stranded DNA binding protein(SSB), or a combination thereof.

In embodiments, the denaturant is a buffered solution including betaine,dimethyl sulfoxide (DMSO), ethylene glycol, formamide, glycerol,guanidine thiocyanate, 4-methylmorpholine 4-oxide (NMO), TMAC, or amixture thereof. In embodiments, the denaturant is a buffered solutionincluding betaine, dimethyl sulfoxide (DMSO), ethylene glycol,formamide, or a mixture thereof.

In embodiments, each invasion-reaction mixture includes a denaturantincluding an SSB, a strand-displacing polymerase, and one or morecrowding agents. In embodiments, the denaturant does not include achemical denaturant (e.g., betaine, DMSO, ethylene glycol, formamide,guanidine thiocyanate, NMO, TMAC, or a mixture thereof). In embodiments,the SSB in the denaturant is T4 gp32 protein, SSB protein, T7 gene 2.5SSB protein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB,Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, orExtreme Thermostable Single-Stranded DNA Binding Protein (ET-SSB). Inembodiments, the strand-displacing polymerase in the denaturant is Bstlarge fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst2.0polymerase, Bsu polymerase, SD polymerase, Vent exo-polymerase, Bsm DNAPolymerase, Phi29 polymerase, or a mutant thereof. In embodiments, thecrowding agent in the denaturant is poly(ethylene glycol) (e.g., PEG200, PEG 600, PEG 800, PEG 2,050, PEG 4,600, PEG 6,000, PEG 8,000, PEG10,000, PEG 20,000, or PEG 35,000). In embodiments, PEG is present inthe denaturant at a concentration of 1% to 25%. In embodiments, PEG ispresent in the denaturant at a concentration of about 1%, about 5%,about 10%, about 15%, about 20%, or about 25%. In embodiments, thedenaturant is a buffered solution including T4 gp32 protein, Bsupolymerase, and 5 to 10% PEG 20,000. In embodiments, the denaturant is abuffered solution including T4 gp32 protein, Bsu polymerase, and 5% PEG20,000. In embodiments, the denaturant is a buffered solution includingT4 gp32 protein, Bsu polymerase, and 10% PEG 20,000.

In embodiments, the SSB is T4 gp32 protein, SSB protein, T7 gene 2.5 SSBprotein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB,Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, orExtreme Thermostable Single-Stranded DNA Binding Protein (ET-SSB). Inembodiments, the SSB is active (i.e., has measurable activity) attemperatures less than about 72° C. In embodiments, the SSB is active(i.e., has measurable activity) at temperatures about 72° C. Inembodiments, the SSB is active (i.e., has measurable activity) attemperatures greater than about 72° C.

In embodiments, the method further includes contacting the invasionprimer with a recombinase, a crowding agent, a loading factor, asingle-stranded binding (SSB) protein, or a combination thereof.

In embodiments, generating the invasion strand includes (i) forming acomplex including a portion of the double-stranded amplificationproduct, an invasion primer, and a homologous recombination complexincluding a recombinase, (ii) releasing the recombinase, and (iii) in aprimer extension reaction, extending the invasion primer with astrand-displacing polymerase. In embodiments, the strand-displacingpolymerase is Bst large fragment (Bst LF) polymerase, Bst 3.0polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase, Ventexo-polymerase, Bsm DNA Polymerase, Phi29 polymerase, or a mutantthereof. In embodiments, the recombinase is a T4 UvsX, RecA, RecT, RecO,or Rad51 protein.

In embodiments, the homologous recombination complex further includes acrowding agent. In embodiments, the crowding agent includespoly(ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), bovine serumalbumin (BSA), dextran, Ficoll (e.g., Ficoll 70 or Ficoll 400),glycerol, or a combination thereof. In embodiments, the crowding agentis poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050,PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000),dextran sulfate, bovine pancreatic trypsin inhibitor (BPTI),ribonuclease A, lysozyme, O-lactoglobulin, hemoglobin, bovine serumalbumin (BSA), or poly(sodium 4-styrene sulfonate) (PSS). Inembodiments, the crowding agent is PEG 200, PEG 600, PEG 800, PEG 2,050,PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000.In embodiments, the crowding agent is PEG 10,000, PEG 20,000, or PEG35,000.

In embodiments, the homologous recombination complex further includes aloading factor, a single-stranded binding (SSB) protein, or both. Inembodiments, the homologous recombination complex includes asingle-stranded binding (SSB) protein. In embodiments, the SSB proteinis T4 gp32 protein, SSB protein, Extreme Thermostable Single-StrandedDNA Binding Protein (ET-SSB), T7 gene 2.5 SSB protein, Thermococcuskodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobussolfataricus (SSO) SSB, or phi29 SSB protein.

In embodiments, the homologous recombination complex further includes aloading factor. In embodiments, the loading factor includes a T4 UvsYprotein.

In embodiments, generating the invasion strand includes thermallycycling between (i) about 72-80° C. for about 5 seconds to about 30seconds (referred to as cycle 1); and (ii) about 60-70° C. for about 30to 90 seconds (referred to as cycle 2). In embodiments, the methodincludes a plurality of thermal cycles in a periodic order (e.g., cycletype 1, cycle 2, cycle 1, etc.). In embodiments, generating the invasionstrand includes thermally cycling between (i) about 67-80° C. for about5 seconds to about 30 seconds (referred to as cycle 1); and (ii) about60-70° C. for about 30 to 90 seconds (referred to as cycle 2). Inembodiments, the method includes a plurality of thermal cycles in aperiodic order (e.g., cycle type 1, cycle 2, cycle 1, etc.).

In embodiments, one or more invasion primers transiently hybridize tothe first or second strand. For example, the denaturing conditions inthe invasion-reaction mix may be too stringent for the invasion primerto fully and stably hybridize for a significant time, however if apolymerase is present in the invasion-reaction mixture, the polymerasecould still extend the invasion primer. In embodiments, generating thefirst invasion strand includes transient hybridization of one or moreinvasion primers to the second strand, and extending the one or moreinvasion strand during their transient hybridization by a polymerase. Inembodiments, the invasion primer partially hybridizes (e.g., less than100% of the invasion primer hybridizes) to the second strand. Inembodiments, the invasion primer hybridizes to the second strand and isextended with a polymerase. In embodiments, the invasion primer does notremain fully annealed to the second strand while the polymerase extendsthe invasion primer. In embodiments, at least three nucleotides of theinvasion primer (e.g., the three nucleotides at the 3′ end of theinvasion primer) hybridize to the second strand, and in the presence ofa strand displacing polymerase the 3′ end of the invasion primer isextended. In embodiments, about 25% to about 90% of the invasion primerhybridizes to the second strand. In embodiments, about 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 90% of theinvasion primer hybridizes to the second strand.

In embodiments, the strand-displacing enzyme is an SD polymerase, Bstlarge fragment polymerase, or a phi29 polymerase or mutant thereof. Inembodiments, the strand-displacing polymerase is phi29 polymerase, phi29mutant polymerase or a thermostable phi29 mutant polymerase. A “phipolymerase” (or “Φ29 polymerase”) is a DNA polymerase from the Φ29 phageor from one of the related phages that, like Φ29, contain a terminalprotein used in the initiation of DNA replication. For example, phi29polymerases include the B103, GA-1, PZA, Φ15, BS32, M2Y (also known asM2), Nf, G1, Cp-1, PRD1, PZE, SFS, Cp-5, Cp-7, PR4, PR5, PR722, L17,Φ21, and AV-1 DNA polymerases, as well as chimeras thereof. A phi29mutant DNA polymerase includes one or more mutations relative tonaturally-occurring wild-type phi29 DNA polymerases, for example, one ormore mutations that alter interaction with and/or incorporation ofnucleotide analogs, increase stability, increase read length, enhanceaccuracy, increase phototolerance, and/or alter another polymeraseproperty, and can include additional alterations or modifications overthe wild-type phi29 DNA polymerase, such as one or more deletions,insertions, and/or fusions of additional peptide or protein sequences.Thermostable phi29 mutant polymerases are known in the art, see forexample US 2014/0322759, which is incorporated herein by reference forall purposes. For example, a thermostable phi29 mutant polymerase refersto an isolated bacteriophage phi29 DNA polymerase including at least onemutation selected from the group consisting of M8R, V51A, M97T, L123S,G197D, K209E, E221K, E239G, Q497P, K512E, E515A, and F526 (relative towild type phi29 polymerase).

In embodiments, the template polynucleotide includes genomic DNA,complementary DNA (cDNA), cell-free DNA (cfDNA), messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free RNA (cfRNA), ornoncoding RNA (ncRNA).

In embodiments, the template polynucleotide is about 100 to 1000nucleotides in length. In embodiments, the template polynucleotide isabout 500 to 2000 nucleotides in length. In embodiments, the templatepolynucleotide is about 1000 to 1000 nucleotides in length. Inembodiments, the template polynucleotide is about 50 to 500 nucleotidesin length. In embodiments, the template polynucleotide is about 500 to1000 nucleotides in length. In embodiments, the template polynucleotideis about 350 nucleotides in length. In embodiments, the templatepolynucleotide is about 10, 20, 50, 100, 150, 200, 300, or 500nucleotides in length. The template polynucleotide molecules can varylength, such as about 100-300 nucleotides long, about 300-500nucleotides long, or about 500-1000 nucleotides long. In embodiments,the template polynucleotide molecular is about 100-1000 nucleotides,about 150-950 nucleotides, about 200-900 nucleotides, about 250-850nucleotides, about 300-800 nucleotides, about 350-750 nucleotides, about400-700 nucleotides, or about 450-650 nucleotides. In embodiments, thetemplate polynucleotide molecule is about 150 nucleotides. Inembodiments, the template polynucleotide is about 100-1000 nucleotideslong. In embodiments, the template polynucleotide is about 100-300nucleotides long. In embodiments, the template polynucleotide is about300-500 nucleotides long. In embodiments, the template polynucleotide isabout 500-1000 nucleotides long. In embodiments, the templatepolynucleotide molecule is about 100 nucleotides. In embodiments, thetemplate polynucleotide molecule is about 300 nucleotides. Inembodiments, the template polynucleotide molecule is about 500nucleotides. In embodiments, the template polynucleotide molecule isabout 1000 nucleotides.

In embodiments the template polynucleotide (e.g., genomic template DNA)is first treated to form single-stranded linear fragments (e.g., rangingin length from about 50 to about 600 nucleotides). Treatment typicallyentails fragmentation, such as by chemical fragmentation, enzymaticfragmentation, or mechanical fragmentation, followed by denaturation toproduce single-stranded DNA fragments. In embodiments, the templatepolynucleotide includes an adapter. The adapter may have otherfunctional elements including tagging sequences (i.e., a barcode),attachment sequences, palindromic sequences, restriction sites,sequencing primer binding sites, functionalization sequences, and thelike. Barcodes can be of any of a variety of lengths. In embodiments,the primer includes a barcode that is 10-50, 20-30, or 4-12 nucleotidesin length. In embodiments, the adapter includes a primer bindingsequence that is complementary to at least a portion of a primer (e.g.,a sequencing primer). Primer binding sites can be of any suitablelength. In embodiments, a primer binding site is about or at least about10, 15, 20, 25, 30, or more nucleotides in length. In embodiments, aprimer binding site is 10-50, 15-30, or 20-25 nucleotides in length.

In embodiments, the template polynucleotide and the double-strandedamplification products include known adapter sequences on the 5′ and 3′ends. In embodiments, the template polynucleotide includes known adaptersequences on the 5′ and 3′ ends. In embodiments, the double-strandedamplification products include known adapter sequences on the 5′ and 3′ends.

In embodiments, prior to hybridizing the invasion primer the methodincludes amplifying the double-stranded polynucleotides with bridgepolymerase chain reaction (bPCR) amplification, solid-phase rollingcircle amplification (RCA), solid-phase exponential rolling circleamplification (eRCA), solid-phase recombinase polymerase amplification(RPA), solid-phase helicase dependent amplification (HDA), templatewalking amplification, or emulsion PCR, or combinations of said methods.In embodiments, generating a double-stranded amplification productincludes bridge polymerase chain reaction (bPCR) amplification,solid-phase rolling circle amplification (RCA), solid-phase exponentialrolling circle amplification (eRCA), solid-phase recombinase polymeraseamplification (RPA), solid-phase helicase dependent amplification (HDA),template walking amplification, or emulsion PCR on particles, orcombinations of the methods. In embodiments, generating adouble-stranded amplification product includes a bridge polymerase chainreaction amplification. In embodiments, generating a double-strandedamplification product includes a thermal bridge polymerase chainreaction (t-bPCR) amplification. In embodiments, generating adouble-stranded amplification product includes a chemical bridgepolymerase chain reaction (c-bPCR) amplification. Chemical bridgepolymerase chain reactions include fluidically cycling a denaturant(e.g., formamide) and maintaining the temperature within a narrowtemperature range (e.g., +/−5° C.). In contrast, thermal bridgepolymerase chain reactions include thermally cycling between hightemperatures (e.g., 85° C.-95° C.) and low temperatures (e.g., 60°C.-70° C.). Thermal bridge polymerase chain reactions may also include adenaturant, typically at a much lower concentration than traditionalchemical bridge polymerase chain reactions.

In embodiments, the solid support includes a plurality ofpolynucleotides, wherein each polynucleotide is attached to the solidsupport at a 5′ end of the polynucleotide.

In embodiments, generating a double-stranded amplification productincludes amplifying the template polynucleotide or complement thereof ona solid support including a plurality of primers attached to the solidsupport, wherein the plurality of primers include a plurality of forwardprimers with complementarity to the template polynucleotide and aplurality of reverse primers with complementarity to a complement of thetemplate polynucleotide, and the amplifying includes a plurality ofcycles of strand denaturation, primer hybridization, and primerextension.

In embodiments, the plurality of strand denaturation cycles aredifferent for one or more cycles, wherein the initial denaturation cycleis maintained at different conditions from the remaining denaturationcycles. For example, in embodiments, the initial denaturation cycle isat about 85° C.-95° C. for about 1 minute to about 10 minutes, whereasdenaturation in the remaining cycles is different (e.g., about 85° C.for about 15-30 sec). In embodiments, the initial denaturation ismaintained at about 85° C.-95° C. for about 5 minutes to about 10minutes. In embodiments, the initial denaturation is maintained at 90°C.-95° C. for about 1 to 10 minutes. In embodiments, the initialdenaturation is maintained at 80° C.-85° C. for about 1 to 10 minutes.In embodiments, the initial denaturation is maintained at 85° C.-90° C.for about 1 to 10 minutes. In embodiments, the initial denaturation ismaintained at about 85° C.-95° C. for about 1 minutes to about 10minutes. In embodiments, the initial denaturation is maintained at about95° C. for about 5 minutes to about 10 minutes. In embodiments, theinitial denaturation is maintained at about 85° C.-95° C. for about 5minutes to about 10 minutes.

In embodiments, generating a double-stranded amplification productincludes a thermal bridge polymerase chain reaction (t-bPCR)amplification. In embodiments, the plurality of cycles includesthermally cycling between (i) about 85° C. for about 15-30 sec fordenaturation, and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation, and (ii) about 65° C. for about 30 secondsfor annealing/extension of the primer.

In embodiments, the plurality of cycles includes thermally cyclingbetween (i) about 80° C. to 90° C. for denaturation, and (ii) about 55°C. to about 65° C. for annealing/extension of the primer. Inembodiments, the plurality of cycles includes thermally cycling between(i) about 85° C. for denaturation, and (ii) about 55° C. forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. fordenaturation, and (ii) about 65° C. for annealing/extension of theprimer. In embodiments, the plurality of cycles includes thermallycycling between (i) less than 80° C. (e.g., 70 to 80° C.) fordenaturation, and (ii) about 55° C. to about 65° C. forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 70° C. fordenaturation, and (ii) about 65° C. for annealing/extension of theprimer. In embodiments, the plurality of cycles includes thermallycycling between (i) about 75° C. for denaturation, and (ii) about 55° C.for annealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. fordenaturation, and (ii) about 65° C. for annealing/extension of theprimer.

In embodiments, the plurality of cycles includes thermally cyclingbetween (i) about 85° C. for less than 1 minute for denaturation, and(ii) about 65° C. for about 1 to 2 minutes for annealing/extension ofthe primer. In embodiments, the plurality of cycles includes thermallycycling between (i) about 85° C. for less than 1 minute fordenaturation, and (ii) about 60° C. to about 65° C. for about 1 minutefor annealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about 30sec for denaturation and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation, and (ii) about 65° C. for about 30 secondsfor annealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation, and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the temperature andduration for the annealing of the primer and the extension of the primerare different. In embodiments, the plurality of cycles includesthermally cycling between (i) about 90° C. to 95° C. for about 15 to 30sec for denaturation and (ii) about 55° C. to about 65° C. for about 30to 60 seconds for annealing and about 65° C. to 70° C. for about 30 to60 seconds for extension of the primer. In embodiments, the plurality ofdenaturation steps is at a temperature of about 80° C.-95° C. Inembodiments, the plurality of denaturation steps is at a temperature ofabout 80° C.-90° C. In embodiments, the plurality of denaturation stepsis at a temperature of about 85° C.-90° C. In embodiments, the pluralityof denaturation steps is at a temperature of about 81° C., 82° C., 83°C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., or about 90° C. Inembodiments, the plurality of denaturation steps is at a temperature ofabout 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., orabout 99° C. In embodiments, the plurality of denaturation steps is at atemperature of about 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93°C., 94° C., or about 95° C. In embodiments, the plurality ofdenaturation steps is at a temperature of about 90° C., 91° C., 92° C.,93° C., 94° C., or about 95° C. In embodiments, the plurality ofdenaturation steps is at a temperature of about 70° C.-85° C. Inembodiments, the plurality of denaturation steps is at a temperature ofabout 70° C.-80° C. In embodiments, the plurality of denaturation stepsis at a temperature of about 75° C.-80° C. In embodiments, the pluralityof denaturation steps is at a temperature of about 70° C., 71° C., 72°C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., or about 80°C. In embodiments, the annealing/extension of the primer cycle is at atemperature of about 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61°C., 62° C., 63° C., 64° C., or about 65° C.

In embodiments, amplifying includes incubation in a denaturant. Inembodiments, the denaturant is acetic acid, ethylene glycol,hydrochloric acid, nitric acid, formamide, guanidine, sodium salicylate,sodium hydroxide, dimethyl sulfoxide (DMSO), propylene glycol, urea, ora mixture thereof. In embodiments, the denaturant is an additive thatlowers a DNA denaturation temperature. In embodiments, the denaturant isbetaine, dimethyl sulfoxide (DMSO), ethylene glycol, formamide,glycerol, guanidine thiocyanate, 4-methylmorpholine 4-oxide (NMO), or amixture thereof. In embodiments, the denaturant is betaine, dimethylsulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidinethiocyanate, or 4-methylmorpholine 4-oxide (NMO).

In embodiments, amplifying includes a plurality of cycles of stranddenaturation, primer hybridization, and primer extension. Although eachcycle will include each of these three events (denaturation,hybridization, and extension), events within a cycle may or may not bediscrete. For example, each step may have different reagents and/orreaction conditions (e.g., temperatures). Alternatively, some steps mayproceed without a change in reaction conditions. For example, extensionmay proceed under the same conditions (e.g., same temperature) ashybridization. After extension, the conditions are changed to start anew cycle with a new denaturation step, thereby amplifying theamplicons. Primer extension products from an earlier cycle may serve astemplates for a later amplification cycle. In embodiments, the pluralityof cycles is about 5 to about 50 cycles. In embodiments, the pluralityof cycles is about 10 to about 45 cycles. In embodiments, the pluralityof cycles is about 10 to about 20 cycles. In embodiments, the pluralityof cycles is about 20 to about 30 cycles. In embodiments, the pluralityof cycles is 10 to 45 cycles. In embodiments, the plurality of cycles is10 to 20 cycles. In embodiments, the plurality of cycles is 20 to 30cycles. In embodiments, the plurality of cycles is about 10 to about 45cycles. In embodiments, the plurality of cycles is about 20 to about 30cycles.

In embodiments, the double-stranded amplification product is provided ina clustered array. In embodiments, the clustered array includes aplurality of double-stranded amplification products localized todiscrete sites on a solid support. In embodiments, the solid support isa bead. In embodiments, the solid support is substantially planar. Inembodiments, the solid support is contained within a flow cell.

In embodiments, the sequencing includes sequencing-by-synthesis,sequencing-by-binding, sequencing by ligation, or pyrosequencing. Inembodiments, generating a first sequencing read or a second sequencingread includes a sequencing by synthesis process. In embodiments,generating a first sequencing read or a second sequencing read includesa sequencing-by-binding. As used herein, “sequencing-by-binding” refersto a sequencing technique wherein specific binding of a polymerase andcognate nucleotide to a primed template nucleic acid molecule (e.g.,blocked primed template nucleic acid molecule) is used for identifyingthe next correct nucleotide to be incorporated into the primer strand ofthe primed template nucleic acid molecule. The specific bindinginteraction need not result in chemical incorporation of the nucleotideinto the primer. In some embodiments, the specific binding interactioncan precede chemical incorporation of the nucleotide into the primerstrand or can precede chemical incorporation of an analogous, nextcorrect nucleotide into the primer. Thus, detection of the next correctnucleotide can take place without incorporation of the next correctnucleotide. As used herein, the “next correct nucleotide” (sometimesreferred to as the “cognate” nucleotide) is the nucleotide having a basecomplementary to the base of the next template nucleotide. The nextcorrect nucleotide will hybridize at the 3′-end of a primer tocomplement the next template nucleotide. The next correct nucleotide canbe, but need not necessarily be, capable of being incorporated at the 3′end of the primer. For example, the next correct nucleotide can be amember of a ternary complex that will complete an incorporation reactionor, alternatively, the next correct nucleotide can be a member of astabilized ternary complex that does not catalyze an incorporationreaction. A nucleotide having a base that is not complementary to thenext template base is referred to as an “incorrect” (or “non-cognate”)nucleotide.

In embodiments, generating a sequencing read includes executing aplurality of sequencing cycles, each cycle including extending thesequencing primer by incorporating a nucleotide or nucleotide analogueusing a polymerase and detecting a characteristic signature indicatingthat the nucleotide or nucleotide analogue has been incorporated. Inembodiments, the method further includes incorporating one or moreunmodified dNTPs or one or more ddNTPs into the 3′ end of the extendedsequencing primer.

In embodiments, the method includes sequencing the first and/or thesecond strand of a double-stranded amplification product by extending asequencing primer hybridized thereto. A variety of sequencingmethodologies can be used such as sequencing-by-synthesis (SBS),pyrosequencing, sequencing by ligation (SBL), or sequencing byhybridization (SBH). Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568;and. 6,274,320, each of which is incorporated herein by reference in itsentirety). In pyrosequencing, released Ppi can be detected by beingconverted to adenosine triphosphate (ATP) by ATP sulfurylase, and thelevel of ATP generated can be detected via light produced by luciferase.In this manner, the sequencing reaction can be monitored via aluminescence detection system. In both SBL and SBH methods, targetnucleic acids, and amplicons thereof, that are present at features of anarray are subjected to repeated cycles of oligonucleotide delivery anddetection. SBL methods, include those described in Shendure et al.Science 309:1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341,each of which is incorporated herein by reference in its entirety; andthe SBH methodologies are as described in Bains et al., Journal ofTheoretical Biology 135(3), 303-7 (1988); Drmanac et al., NatureBiotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773(1995); and WO 1989/10977, each of which is incorporated herein byreference in its entirety.

In SBS, extension of a nucleic acid primer along a nucleic acid templateis monitored to determine the sequence of nucleotides in the template.The underlying chemical process can be catalyzed by a polymerase,wherein fluorescently labeled nucleotides are added to a primer (therebyextending the primer) in a template dependent fashion such thatdetection of the order and type of nucleotides added to the primer canbe used to determine the sequence of the template. A plurality ofdifferent nucleic acid fragments that have been attached at differentlocations of an array can be subjected to an SBS technique underconditions where events occurring for different templates can bedistinguished due to their location in the array. In embodiments, thesequencing step includes annealing and extending a sequencing primer toincorporate a detectable label that indicates the identity of anucleotide in the target polynucleotide, detecting the detectable label,and repeating the extending and detecting steps. In embodiments, themethods include sequencing one or more bases of a target nucleic acid byextending a sequencing primer hybridized to a target nucleic acid (e.g.,an amplification product produced by the amplification methods describedherein). In embodiments, the sequencing step may be accomplished by asequencing-by-synthesis (SBS) process. In embodiments, sequencingcomprises a sequencing by synthesis process, where individualnucleotides are identified iteratively, as they are polymerized to forma growing complementary strand. In embodiments, nucleotides added to agrowing complementary strand include both a label and a reversible chainterminator that prevents further extension, such that the nucleotide maybe identified by the label before removing the terminator to add andidentify a further nucleotide. Such reversible chain terminators includeremovable 3′ blocking groups, for example as described in U.S. Pat. Nos.10,738,072, 7,541,444 and 7,057,026. Once such a modified nucleotide hasbeen incorporated into the growing polynucleotide chain complementary tothe region of the template being sequenced, there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase cannot add further nucleotides. Once the identity of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides it ispossible to deduce the DNA sequence of the DNA template. Non-limitingexamples of suitable labels are described in U.S. Pat. Nos. 8,178,360,5,188,934 (4,7-dichlorofluorscein dyes); U.S. Pat. No. 5,366,860(spectrally resolvable rhodamine dyes); U.S. Pat. No. 5,847,162(4,7-dichlororhodamine dyes); U.S. Pat. No. 4,318,846 (ether-substitutedfluorescein dyes); U.S. Pat. No. 5,800,996 (energy transfer dyes); U.S.Pat. No. 5,066,580 (xanthene dyes): U.S. Pat. No. 5,688,648 (energytransfer dyes); and the like.

Sequencing includes, for example, detecting a sequence of signals.Examples of sequencing include, but are not limited to, sequencing bysynthesis (SBS) processes in which reversibly terminated nucleotidescarrying fluorescent dyes are incorporated into a growing strand,complementary to the target strand being sequenced. In embodiments, thenucleotides are labeled with up to four unique fluorescent dyes. Inembodiments, the nucleotides are labeled with at least two uniquefluorescent dyes. In embodiments, the readout is accomplished byepifluorescence imaging. A variety of sequencing chemistries areavailable, non-limiting examples of which are described herein.

Flow cells provide a convenient format for housing an array of clustersproduced by the methods described herein, in particular when subjectedto an SBS or other detection technique that involves repeated deliveryof reagents in cycles. For example, to initiate a first SBS cycle, oneor more labeled nucleotides and a DNA polymerase in a buffer, can beflowed into/through a flow cell that houses an array of clusters. Theclusters of an array where primer extension causes a labeled nucleotideto be incorporated can then be detected. Optionally, the nucleotides canfurther include a reversible termination moiety that temporarily haltsfurther primer extension once a nucleotide has been added to a primer.For example, a nucleotide analog having a reversible terminator moietycan be added to a primer such that subsequent extension cannot occuruntil a deblocking agent (e.g., a reducing agent) is delivered to removethe moiety. Thus, for embodiments that use reversible termination, adeblocking reagent (e.g., a reducing agent) can be delivered to the flowcell (before, during, or after detection occurs). Washes can be carriedout between the various delivery steps as needed. The cycle can then berepeated N times to extend the primer by N nucleotides, therebydetecting a sequence of length N. Example SBS procedures, fluidicsystems and detection platforms that can be readily adapted for use withan array produced by the methods of the present disclosure aredescribed, for example, in Bentley et al., Nature 456:53-59 (2008), USPatent Publication 2018/0274024, WO 2017/205336, US Patent Publication2018/0258472, each of which are incorporated herein in their entiretyfor all purposes.

Use of the sequencing method outlined above is a non-limiting example,as essentially any sequencing methodology which relies on successiveincorporation of nucleotides into a polynucleotide chain can be used.Suitable alternative techniques include, for example, pyrosequencingmethods, FISSEQ (fluorescent in situ sequencing), MPSS (massivelyparallel signature sequencing), or sequencing by ligation-based methods.

In embodiments, generating a sequencing read includes determining theidentity of the nucleotides in the template polynucleotide (orcomplement thereof). In embodiments, a sequencing read, e.g., a firstsequencing read or a second sequencing read, includes determining theidentity of a portion (e.g., 1, 2, 5, 10, 20, 50 nucleotides) of thetotal template polynucleotide. In embodiments the first sequencing readdetermines the identity of 5-10 nucleotides and the second sequencingread determines the identity of more than 5-10 nucleotides (e.g., 11 to200 nucleotides). In embodiments the first sequencing read determinesthe identity of more than 5-10 nucleotides (e.g., 11 to 200 nucleotides)and the second sequencing read determines the identity of 5-10nucleotides. In embodiments, following the generation of a sequencingread, subsequent extension is performed using a plurality of standard(e.g., non-modified) dNTPs until the complementary strand is copied. Inother embodiments, following the generation of a sequencing read,subsequent extension is performed using a plurality of dideoxynucleotide triphosphates (ddNTPs) to prevent further extension of thefirst sequencing read product during a second sequencing read. Inembodiments, following the identification of at least 5-10 (e.g., 11 to200 nucleotides, or up to 1000 nucleotides), subsequent extension isperformed using a plurality of standard (e.g., non-modified) dNTPs untilthe complementary strand is copied. In embodiments, following theidentification of at least 5-10 (e.g., 11 to 200 nucleotides, or up to1000 nucleotides), subsequent extension is performed using a pluralityof dideoxy nucleotide triphosphates (ddNTPs) to prevent furtherextension of the sequencing read product.

In embodiments, following the generation of a first sequencing read, thecleavable site located within the invasion primer is cleaved, therebyexposing a free 5′ phosphate in the invasion strand. In embodiments,following the generation of a first sequencing read, the cleavable sitelocated within the invasion primer is cleaved with a cleaving agent,thereby exposing a free 5′ phosphate in the invasion strand, and theinvasion strand is removed (e.g., enzymatically digested using anexonuclease enzyme). In embodiments, following generation of a firstsequencing read, the invasion strand is cleaved at one or more cleavablesites. In embodiments, following cleavage at one or more cleavablesites, the extension product of the invasion primer (i.e., the invasionstrand) is removed under suitable non-aggressive conditions (e.g.,degraded or denatured under conditions that leave the complementarystrand intact, and optionally still hybridized to at least a portion ofthe invasion primer). In embodiments, the cleavable site is a dU. Inembodiments, the cleaving agent includes a glycosylase and one or moresuitable endonucleases. In embodiments, cleavage is performed underalkaline (e.g., pH greater than 8) buffer conditions at between 40° C.to 80° C. In embodiments, degradation of the invasion strand isenzymatic degradation. In embodiments, degradation of the invasionstrand is accomplished with a 5′ to 3′ exonuclease. In embodiments, the5′ to 3′ exonuclease is lambda exonuclease, or a mutant thereof. Inembodiments, following the degradation of the invasion strand, thecleaved invasion primer subsequently initiates a second sequencing read.In embodiments, the second sequencing read is generated without removalof the first sequencing read. In embodiments, the invasion primer (or aportion thereof) is the sequencing primer.

In embodiments, the sequencing method relies on the use of modifiednucleotides that can act as reversible reaction terminators. Once themodified nucleotide has been incorporated into the growingpolynucleotide chain complementary to the region of the template beingsequenced there is no free 3′-OH group available to direct furthersequence extension and therefore the polymerase cannot add furthernucleotides. Once the identity of the base incorporated into the growingchain has been determined, the 3′ reversible terminator may be removedto allow addition of the next successive nucleotide. These suchreactions can be done in a single experiment if each of the modifiednucleotides has attached a different label, known to correspond to theparticular base, to facilitate discrimination between the bases added ateach incorporation step. Alternatively, a separate reaction may becarried out containing each of the modified nucleotides separately.

In embodiments, the method further includes terminating extension byincorporating one or more unmodified dNTPs and/or one or more ddNTPsinto the 3′ end of the extension strand and hybridizing a secondsequencing primer to the second strand and incorporating one or morenucleotides into the second sequencing primer with a polymerase tocreate a second extension strand; and detecting the one or moreincorporated nucleotides so as to identify each incorporated nucleotidein said second extension strand. In embodiments, the method furtherincludes terminating extension by incorporating one or more unmodifieddNTPs or one or more ddNTPs into the 3′ end of the second extensionstrand; removing the invasion strand; hybridizing a third sequencingprimer to the first strand and incorporating one or more nucleotidesinto the third sequencing primer with a polymerase to create a thirdextension strand; and detecting the one or more incorporated nucleotidesso as to identify each incorporated nucleotide in said third extensionstrand. In embodiments, the method includes terminating extension byincorporating one or more unmodified dNTPs or one or more ddNTPs intothe 3′ end of the third extension strand; and hybridizing a fourthsequencing primer to the first strand and incorporating one or morenucleotides into the fourth sequencing primer with a polymerase tocreate a fourth extension strand; and detecting the one or moreincorporated nucleotides so as to identify each incorporated nucleotidein said fourth extension strand. In embodiments, the method furtherincludes terminating extension by incorporating one or more unmodifieddNTPs and/or one or more ddNTPs into the 3′ end of the extension strand;hybridizing a second sequencing primer to the second strand andincorporating one or more nucleotides into the second sequencing primerwith a polymerase to create a second extension strand; and detecting theone or more incorporated nucleotides so as to identify each incorporatednucleotide in said second extension strand; terminating extension byincorporating one or more unmodified dNTPs or one or more ddNTPs intothe 3′ end of the second extension strand; removing the invasion strand;hybridizing a third sequencing primer to the first strand andincorporating one or more nucleotides into the third sequencing primerwith a polymerase to create a third extension strand; and detecting theone or more incorporated nucleotides so as to identify each incorporatednucleotide in said third extension strand; terminating extension byincorporating one or more unmodified dNTPs or one or more ddNTPs intothe 3′ end of the third extension strand; and hybridizing a fourthsequencing primer to the first strand and incorporating one or morenucleotides into the fourth sequencing primer with a polymerase tocreate a fourth extension strand; and detecting the one or moreincorporated nucleotides so as to identify each incorporated nucleotidein said fourth extension strand.

In embodiments, the method further includes terminating extension byincorporating one or more unmodified dNTPs and/or one or more ddNTPsinto the 3′ end of the extension strand. In embodiments, the methodfurther includes terminating extension by incorporating one or moreunmodified dNTPs. In embodiments, the method further includesterminating extension by incorporating one or more ddNTPs into the 3′end of the extension strand.

In embodiments, the method further includes hybridizing a secondsequencing primer to the second strand and incorporating one or morenucleotides (e.g., labeled nucleotides) with a polymerase into thesecond sequencing primer to create a second extension strand; anddetecting the one or more incorporated nucleotides so as to identifyeach incorporated nucleotide in said second extension strand. Inembodiments, the nucleotides are modified nucleotides including a labeland a reversible terminator, as described herein.

The modified nucleotides may carry a label (e.g., a fluorescent label)to facilitate their detection. Each nucleotide type may carry adifferent fluorescent label. However, the detectable label need not be afluorescent label. Any label can be used which allows the detection ofan incorporated nucleotide. One method for detecting fluorescentlylabeled nucleotides includes using laser light of a wavelength specificfor the labeled nucleotides, or the use of other suitable sources ofillumination. The fluorescence from the label on the nucleotide may bedetected (e.g., by a CCD camera, CMOS camera, or other suitabledetection means).

In embodiments, the methods of sequencing a nucleic acid includeextending a complementary polynucleotide (e.g., a primer) that ishybridized to the nucleic acid by incorporating a first nucleotide. Inembodiments, the method includes a buffer exchange or wash step (e.g.,between each sequencing cycle). In embodiments, the methods ofsequencing a nucleic acid include a sequencing solution. The sequencingsolution includes (a) an adenine nucleotide, or analog thereof, (b) (i)a thymine nucleotide, or analog thereof, or (ii) a uracil nucleotide, oranalog thereof, (c) a cytosine nucleotide, or analog thereof, and (d) aguanine nucleotide, or analog thereof.

In certain embodiments, the sequencing methods provided herein comprisessequencing both strands of a double-stranded nucleic acid with an errorrate of 5×10⁻⁵ or less, 1×10⁻⁵ or less, 5×10⁻⁶ or less, 1×10⁻⁶ or less,5×10⁻⁷ or less, 1×10⁻⁷ or less, 5×10⁻⁸ or less, or 1×10⁻⁸ or less. Incertain embodiments, the sequencing methods provided herein comprisessequencing both strands of a double-stranded nucleic acid with an errorrate of 5×10⁻⁵ to 1×10⁻⁸, 1×10⁻⁵ to 1×10⁻⁸, 5×10⁻⁵ to 1×10⁻⁷, 1×10⁻⁵ to1×10⁻⁷, 5×10⁻⁶, to 1×10⁻⁸, or 1×10⁻⁶ to 1×10⁻⁸. In certain embodiments,the sequencing methods provided herein comprises sequencing both strandsof a double-stranded nucleic acid with an error rate of 1×10⁻⁷ to1×10⁻⁸.

In an aspect is provided a method of reducing GC bias in a plurality ofsequencing reads, the method including sequencing a templatepolynucleotide to generate a plurality of sequencing reads as describedherein. In embodiments, the method includes: generating adouble-stranded amplification product including a first strandhybridized to a second strand, wherein (i) the double-strandedamplification product includes the template polynucleotide or complementthereof, and (ii) the first strand and second strand are both attachedto a solid support; generating a first invasion strand hybridized to thesecond strand by hybridizing one or more invasion primers to the secondstrand, as described herein, for example wherein generating the firstinvasion strand includes a first plurality of invasion-primer extensioncycles followed by a second plurality of invasion-primer extensioncycles, wherein the reaction conditions for the first plurality ofinvasion-primer extension cycles are different than the second pluralityof invasion-primer extension cycles and extending the one or moreinvasion primers; generating a first sequencing read by hybridizing oneor more sequencing primers to the first strand, and extending the one ormore first sequencing primers. In embodiments, the invasion primer isnot covalently attached to the solid support.

In embodiments, the method includes: generating a double-strandedamplification product including a first strand hybridized to a secondstrand, wherein (i) the double-stranded amplification product includesthe template polynucleotide or complement thereof, and (ii) the firststrand and second strand are both attached to a solid support;generating a first invasion strand hybridized to the second strand byhybridizing one or more invasion primers to the second strand, whereingenerating the invasion strand comprises alternating between a firstplurality of invasion-primer extension cycles and a second plurality ofinvasion-primer extension cycles, wherein the reaction conditions forthe first plurality of invasion-primer extension cycles are differentthan the second plurality of invasion-primer extension cycles andextending the one or more invasion primers; generating a firstsequencing read by hybridizing one or more sequencing primers to thefirst strand, and extending the one or more first sequencing primers.

In embodiments, generating the invasion strand includes a firstplurality of invasion-primer extension cycles followed by a secondplurality of invasion-primer extension cycles, wherein the reactionconditions for the first plurality of invasion-primer extension cyclesare different than the second plurality of invasion-primer extensioncycles. In embodiments, the method further includes a third plurality ofinvasion-primer extension cycles, wherein the reaction conditions forthe third plurality of invasion-primer extension cycles are optionallydifferent than the first or second plurality of invasion-primerextension cycles. In embodiments, the method further includes a thirdplurality of invasion-primer extension cycles, wherein the reactionconditions for the third plurality of invasion-primer extension cyclesare the same as the first plurality of invasion-primer extension cycles.

In an aspect is provided a method of generating a template for nucleicacid sequencing reaction. In embodiments, the method includes providinga solid support including a plurality of immobilized oligonucleotideprimers attached to the solid support via a linker, wherein theplurality of oligonucleotide primers include a plurality of forwardprimers and a plurality of reverse primers, amplifying a templatenucleic acid by using the oligonucleotide primers attached to the solidsupport to generate a plurality of double-stranded amplificationproducts, each double-stranded amplification product including a firststrand hybridized to a second strand, wherein (i) each double-strandedamplification product includes the template polynucleotide or complementthereof, and (ii) the first strand and second strand are both attachedto the solid support; and generating a first invasion strand hybridizedto the second strand by hybridizing one or more invasion primers to thesecond strand, and extending the one or more invasion primers; therebygenerating a template nucleic acid for a nucleic acid sequencingreaction. In embodiments, the method further includes hybridizing one ormore sequencing primers to the first strand. In embodiments, the methodincludes generating a cluster of ssDNA templates. In embodiments, theinvasion primer is not covalently attached to the solid support. Inembodiments, the invasion strand is not covalently attached to the solidsupport.

In another aspect is provided a method including: amplifying a templatenucleic acid by contacting the template nucleic acid with a plurality ofoligonucleotide primers attached to a solid support to generate aplurality of double-stranded amplification products, eachdouble-stranded amplification product including a first strandhybridized to a second strand, wherein the first strand and secondstrand are both attached to the solid support; and generating a firstinvasion strand hybridized to the second strand by hybridizing one ormore invasion primers to the second strand, and extending the one ormore invasion primers to produce a single-stranded first strand. Inembodiments, the invasion primer is not covalently attached to the solidsupport.

In an aspect is provided a method of removing a polynucleotidehybridized to a first strand, wherein the polynucleotide includes one ormore of cleavable sites. In embodiments, the method includes fragmentinga polynucleotide in the presence of a plurality of dsDNApolynucleotides. In embodiments, the method includes contacting thepolynucleotide with a cleaving agent thereby fragmenting thepolynucleotide and generating two or more fragments. In embodiments, themethod includes denaturing the fragments (e.g., contacting the fragmentswith a chemical denaturant, increasing the temperature, or a combinationthereof). In embodiments, the method includes digesting the fragments(e.g., contacting the fragments with one or more exonuclease enzymes).In embodiments, the method includes modulating the temperature to be ator below the calculated or predicted melting temperature (Tm) of thefragments (e.g., about 0° C. to about 65° C.). In embodiments, themethod includes modulating the temperature to be at about 50° C. toabout 65° C.

In embodiments, the first strand is covalently attached to a solidsupport. In embodiments, the polynucleotide, alternatively referred toherein as the third polynucleotide and/or the invasion primer, is notattached to a solid support. In embodiments, the first strand isattached to a solid support, wherein the solid support includes aplurality of double-stranded polynucleotides. In embodiments, the firststrand is in a colony of double-stranded polynucleotides. Inembodiments, the solid support includes a second strand hybridized to asequenced strand, wherein the sequenced strand includes one or moresequenced nucleotides. In embodiments, the sequenced nucleotides includea scar remnant (e.g., an alkynyl moiety attached to the nucleobase). Inembodiments, the nucleotides have the formula:

wherein B is a nucleobase, R¹ is the scar remnant, and “

” is the attachment point to the remainder of the sequenced strandpolynucleotide.

In embodiments, B is a divalent nucleobase. In embodiments, B is

In embodiments, B is

In embodiments, R¹ is hydrogen, —OH, —NH, a substituted or unsubstitutedalkyl or substituted or unsubstituted heteroalkyl. In embodiments, R¹ ishydrogen. In embodiments, R¹ is —OH. In embodiments, R¹ is —NH. Inembodiments, R¹ is a substituted or unsubstituted alkyl or substitutedor unsubstituted heteroalkyl. In embodiments, R¹ is a substituted orunsubstituted alkenyl. In embodiments, R¹ is a substituted orunsubstituted alkynyl. In embodiments, R¹ is a substituted orunsubstituted heteroalkenyl. In embodiments, R¹ is a substituted orunsubstituted heteroalkynyl. In embodiments, R¹ is a substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl. In embodiments,R¹ is substituted with an oxo or —OH. In embodiments, R¹ is substitutedwith an oxo and —OH.

In embodiments, R¹ is an oxo-substituted heteroalkyl (e.g., 2 to 10membered heteroalkyl, 2 to 8 membered heteroalkyl, or 4 to 8 memberedheteroalkyl). In embodiments, R¹ is an oxo-substituted heteroalkenyl(e.g., 2 to 10 membered heteroalkenyl, 2 to 8 membered heteroalkenyl, or4 to 8 membered heteroalkenyl). In embodiments, R¹ is an oxo-substitutedheteroalkynyl (e.g., 2 to 10 membered heteroalkynyl, 2 to 8 memberedheteroalkynyl, or 4 to 8 membered heteroalkynyl). In embodiments, R¹ isan oxo-substituted 10 membered heteroalkynyl. In embodiments, R¹ is anoxo-substituted 9 membered heteroalkynyl. In embodiments, R¹ is anoxo-substituted 8 membered heteroalkynyl. In embodiments, R¹ is anoxo-substituted 7 membered heteroalkynyl. In embodiments, R¹ is anoxo-substituted 6 membered heteroalkynyl.

In embodiments, the one or more nucleotides including a scar remnantinclude a nucleobase having the formula

In embodiments, the one or more nucleotides including a scar remnantinclude a nucleobase having the formula

In embodiments, the calculated or predicted melting temperature (Tm) ofthe fragments is about 50° C. to about 75° C. In embodiments, thecalculated or predicted melting temperature (Tm) of the fragments isabout 60° C. to about 75° C. In embodiments, the calculated or predictedmelting temperature (Tm) of the fragments is about 50° C. to about 65°C. In embodiments, the calculated or predicted melting temperature (Tm)of the fragments is less than about 75° C. In embodiments, thecalculated or predicted melting temperature (Tm) of the fragments isless than about 65° C. In embodiments, the calculated or predictedmelting temperature (Tm) of the fragments is less than about 60° C. Inembodiments, two or more fragments are generated. In embodiments, threeor more fragments are generated. In embodiments, four or more fragmentsare generated. In embodiments, at least three fragments are generated.In embodiments, four fragments are generated.

In embodiments, the fragments are 3-10 nucleotides in length. Inembodiments, the fragments are 3-15 nucleotides in length. Inembodiments, the fragments are 5 to 20 nucleotides in length. Inembodiments, the fragments are 4 to 6 nucleotides in length.

P-EMBODIMENTS

The present disclosure provides the following illustrative embodiments.

Embodiment P1. A method of sequencing a template polynucleotide, themethod comprising: (A) generating a double-stranded amplificationproduct comprising a first strand hybridized to a second strand, wherein(i) the double-stranded amplification product comprises the templatepolynucleotide or complement thereof, and (ii) the first strand andsecond strand are both attached to a solid support; (B) generating afirst invasion strand hybridized to the second strand by hybridizing aninvasion primer to the second strand, and extending the invasion primer,wherein the invasion primer is not covalently attached to the solidsupport; and (C) generating a first sequencing read by hybridizing oneor more sequencing primers to the first strand, and extending the one ormore first sequencing primers.

Embodiment P2. The method of Embodiment P1, wherein the first strand iscovalently attached to the solid support via a first linker and thesecond strand is covalently attached to the solid support via a secondlinker.

Embodiment P3. The method of Embodiment P1 or Embodiment P2, furthercomprising removing the first invasion strand; generating a secondinvasion strand hybridized to the first strand by hybridizing a secondinvasion primer to the first strand, and extending the second invasionprimer, wherein the second invasion primer is not covalently attached tothe solid support; and generating a second sequencing read byhybridizing one or more second sequencing primers to the second strand,and extending the one or more second sequencing primers.

Embodiment P4. The method of Embodiment P1 or Embodiment P2, wherein theinvasion primer comprises a cleavable site.

Embodiment P5. The method of Embodiment P4, wherein the cleavable siteis located at the 3′ end of the invasion primer.

Embodiment P6. The method of Embodiment P4 or Embodiment P5, furthercomprising cleaving the cleavable site in the invasion primer togenerate a free 3′ end within the invasion primer, removing the invasionstrand, and generating a second sequencing read by extending theinvasion primer.

Embodiment P7. The method of Embodiment P1 or Embodiment P2, furthercomprising removing the first strand by cleaving the first strand at acleavable site and generating a second sequencing read by hybridizingone or more second sequencing primers to the second strand; andextending the one or more second sequencing primers.

Embodiment P8. The method of Embodiment P7, wherein the first strandcomprises at least one cleavable site or the first linker comprises atleast one cleavable site.

Embodiment P9. The method of Embodiment P7 or Embodiment P8, whereincleaving comprises enzymatically cleaving the first strand at the atleast one cleavable site.

Embodiment P10. The method of Embodiment P7 or Embodiment P8, whereincleaving comprises chemically cleaving the first strand at the at leastone cleavable site.

Embodiment P11. The method of any one of Embodiment P4 to EmbodimentP10, wherein the cleavable site comprises a diol linker, disulfidelinker, photocleavable linker, abasic site, deoxyuracil triphosphate(dUTP), deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylatednucleotide, ribonucleotide, or a sequence containing a modified orunmodified nucleotide that is specifically recognized by a cleavingagent.

Embodiment P12. The method of any one of Embodiment P4 to EmbodimentP10, wherein the cleavable site comprises one or more ribonucleotides.

Embodiment P13. The method of any one of Embodiment P4 to EmbodimentP10, wherein the cleavable site comprises deoxyuracil triphosphate(dUTP) or deoxy-8-oxo-guanine triphosphate (d-8-oxoG).

Embodiment P14. The method of any one of Embodiment P4 to EmbodimentP10, wherein the first linker comprises a diol linker, disulfide linker,photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP),deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide,ribonucleotide, or a sequence containing a modified or unmodifiednucleotide that is specifically recognized by a cleaving agent.

Embodiment P15. The method of any one of Embodiment P4 to EmbodimentP10, wherein the first strand comprises a diol linker, disulfide linker,photocleavable linker, abasic site, deoxyuracil triphosphate (dUTP),deoxy-8-Oxo-guanine triphosphate (d-8-oxoG), methylated nucleotide,ribonucleotide, or a sequence containing a modified or unmodifiednucleotide that is specifically recognized by a cleaving agent.

Embodiment P16. The method of any one of Embodiment P7 to EmbodimentP15, wherein cleaving the first strand comprises contacting thecleavable site with a cleaving agent, wherein the cleaving agentcomprises a reducing agent, sodium periodate, Rnase, FormamidopyrimidineDNA Glycosylase (Fpg), endonuclease, or uracil DNA glycosylase (UDG).

Embodiment P17. The method of any one of Embodiment P1 to EmbodimentP16, wherein the invasion primer comprises locked nucleic acids (LNAs),Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids(TINAs), bridged nucleic acids (BNAs), 2′-O-methyl RNA:DNA chimericnucleic acids, minor groove binder (MGB) nucleic acids, morpholinonucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleicacids (PNAs), phosphorothioate nucleotides, or combinations thereof.

Embodiment P18. The method of any one of Embodiment P1 to EmbodimentP16, wherein the invasion primer comprises locked nucleic acids (LNAs),Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids(TINAs), bridged nucleic acids (BNAs), peptide nucleic acids (PNAs), orcombinations thereof.

Embodiment P19. The method of any one of Embodiment P1 to EmbodimentP16, wherein the invasion primer comprises locked nucleic acids (LNAs),or peptide nucleic acids (PNAs).

Embodiment P20. The method of any one of Embodiment P1 to EmbodimentP16, wherein generating the first or second invasion strand comprises(i) forming a complex comprising a portion of the double-strandedamplification product, an invasion primer, and a homologousrecombination complex comprising a recombinase, (ii) releasing therecombinase, and (iii) in a primer extension reaction, extending theinvasion primer with a strand-displacing polymerase.

Embodiment P21. The method of Embodiment P20, wherein thestrand-displacing polymerase is Bst large fragment (Bst LF) polymerase,Bst 3.0 polymerase, Bst2.0 polymerase, Bsu polymerase, SD polymerase,Vent exo-polymerase, Phi29 polymerase, or a mutant thereof.

Embodiment P22. The method of Embodiment P20 or Embodiment P21, whereinthe recombinase is a T4 UvsX, RecA, RecT, RecO, or Rad51 protein.

Embodiment P23. The method of any one of Embodiment P20 to EmbodimentP22, wherein the homologous recombination complex further comprises acrowding agent.

Embodiment P24. The method of Embodiment P23, wherein the crowding agentcomprises PEG, PVP, BSA, dextran, Ficoll, glycerol, or a combinationthereof.

Embodiment P25. The method of any one of Embodiment P20 to EmbodimentP22, wherein the homologous recombination complex further comprises aloading factor, a single-stranded binding (SSB) protein, or both.

Embodiment P26. The method of Embodiment P25, wherein the SSB protein isT4 gp32 protein, SSB protein, Extreme Thermostable Single-Stranded DNABinding Protein (ET-SSB), T7 gene 2.5 SSB protein, Thermococcuskodakarensis (KOD) SSB, Thermus thermophilus (TTH) SSB, Sulfolobussolfataricus (SSO) SSB, or phi29 SSB protein.

Embodiment P27. The method of Embodiment P25, wherein the loading factorcomprises a T4 UvsY protein.

Embodiment P28. The method of any one of Embodiment P1 to EmbodimentP27, wherein the invasion primer is about 10 to 100 nucleotides inlength.

Embodiment P29. The method of any one of Embodiment P1 to EmbodimentP27, wherein the invasion primer is about 15 to about 75 nucleotides inlength.

Embodiment P30. The method of any one of Embodiment P1 to EmbodimentP27, wherein the invasion primer is about 10 to about 20 nucleotides inlength.

Embodiment P31. The method of any one of Embodiment P1 to EmbodimentP30, wherein generating the invasion strand comprises a plurality ofinvasion primer extension cycles.

Embodiment P32. The method of any one of Embodiment P1 to EmbodimentP30, wherein generating the invasion strand comprises extending theinvasion primer by incorporating one or more nucleotides using Bst largefragment (Bst LF) polymerase, Bst2.0 polymerase, Bsu polymerase, SDpolymerase, Vent exo-polymerase, Phi29 polymerase, or a mutant thereof.

Embodiment P33. The method of any one of Embodiment P1 to EmbodimentP30, wherein generating the invasion strand comprises contacting thedouble-stranded amplification product with one or more invasion-reactionmixtures; each of said invasion-reaction mixture comprising a pluralityof invasion primers, a plurality of deoxyribonucleotide triphosphate(dNTPs), and a polymerase.

Embodiment P34. The method of Embodiment P33, wherein the polymerase isa strand-displacing polymerase.

Embodiment P35. The method of Embodiment P33, wherein eachinvasion-reaction mixture further comprises a denaturant,single-stranded DNA binding protein (SSB), or a combination thereof.

Embodiment P36. The method of Embodiment P33, wherein eachinvasion-reaction mixture comprises a different amount of a denaturant,single-stranded DNA binding protein (SSB), or a combination thereof.

Embodiment P37. The method of Embodiment P35 or Embodiment P36, whereinthe denaturant is a buffered solution comprising betaine, dimethylsulfoxide (DMSO), ethylene glycol, formamide, glycerol, guanidinethiocyanate, 4-methylmorpholine 4-oxide (NMO), or a mixture thereof.

Embodiment P38. The method of Embodiment P35 or Embodiment P36, whereinthe denaturant is a buffered solution comprising betaine, dimethylsulfoxide (DMSO), ethylene glycol, formamide, or a mixture thereof.

Embodiment P39. The method of any one of Embodiment P35 to EmbodimentP38, wherein the SSB is T4 gp32 protein, SSB protein, T7 gene 2.5 SSBprotein, or phi29 SSB protein, Thermococcus kodakarensis (KOD) SSB,Thermus thermophilus (TTH) SSB, Sulfolobus solfataricus (SSO) SSB, orExtreme Thermostable Single-Stranded DNA Binding Protein (ET-SSB).

Embodiment P40. The method of any one of Embodiment P1 to EmbodimentP30, wherein generating the first or second invasion strand comprises afirst plurality of invasion-primer extension cycles followed by a secondplurality of invasion-primer extension cycles, wherein the reactionconditions for the first plurality of invasion-primer extension cyclesare different than the second plurality of invasion-primer extensioncycles.

Embodiment P41. The method of any one of Embodiment P1 to EmbodimentP30, wherein generating the first or second invasion strand comprisesalternating between a first plurality of invasion-primer extensioncycles and a second plurality of invasion-primer extension cycles,wherein the reaction conditions for the first plurality ofinvasion-primer extension cycles are different than the second pluralityof invasion-primer extension cycles.

Embodiment P42. The method of Embodiment P40 or Embodiment P41, whereinthe reaction conditions for the first plurality of invasion-primerextension cycles comprise higher stringency hybridization conditionsrelative to the second plurality of invasion-primer extension cycles.

Embodiment P43. The method of Embodiment P40 or Embodiment P41, whereinthe reaction conditions for the first plurality of invasion-primerextension cycles comprise incubation in a first denaturant, wherein thefirst denaturant is a buffered solution comprising about 0% to about 50%dimethyl sulfoxide (DMSO); about 0% to about 50% ethylene glycol; about0% to about 20% formamide; or about 0 to about 3M betaine, or a mixturethereof.

Embodiment P44. The method of Embodiment P40 or Embodiment P41, whereinthe reaction conditions for the first plurality of invasion-primerextension cycles comprise incubation in a first denaturant, wherein thefirst denaturant is a buffered solution comprising about 15% to about50% dimethyl sulfoxide (DMSO); about 15% to about 50% ethylene glycol;about 10% to about 20% formamide; or about 0 to about 3M betaine, or amixture thereof.

Embodiment P45. The method of Embodiment P40 or Embodiment P41, whereinthe reaction conditions for the second plurality of invasion-primerextension cycles comprise incubation in a second denaturant, wherein thesecond denaturant is a buffered solution comprising about 0 to about 50%dimethyl sulfoxide (DMSO); about 0 to about 50% ethylene glycol; about 0to about 20% formamide; or about 0 to about 3M betaine, or a mixturethereof.

Embodiment P46. The method of Embodiment P40 or Embodiment P41, whereinthe reaction conditions for the second plurality of invasion-primerextension cycles comprise incubation in a second denaturant, wherein thesecond denaturant is a buffered solution comprising about 0% to about15% dimethyl sulfoxide (DMSO); about 0 to about 15% ethylene glycol;about 0 to about 10% formamide; or about 0 to about 3M betaine, or amixture thereof.

Embodiment P47. The method of Embodiment P45, wherein the firstdenaturant is a buffered solution comprising dimethyl sulfoxide (DMSO);and the second denaturant is a buffered solution comprising dimethylsulfoxide (DMSO) and betaine.

Embodiment P48. The method of Embodiment P45, wherein the firstdenaturant is a buffered solution comprising about 25 to about 35% DMSO;and the second denaturant is a buffered solution comprising about 0 toabout 10% DMSO and about 1M to about 4M betaine.

Embodiment P49. The method of Embodiment P45, wherein the firstdenaturant is a buffered solution comprising about 30% DMSO; and thesecond denaturant is a buffered solution comprising about 5% DMSO, about2.5M betaine.

Embodiment P50. The method of any one of Embodiment P45 to EmbodimentP49, wherein the reaction conditions for the second plurality ofinvasion-primer extension cycles further comprises incubation with a SSBprotein.

Embodiment P51. The method of any one of Embodiment P1 to EmbodimentP30, wherein generating the first or second invasion strand comprisescontacting the double-stranded amplification product with a firstinvasion-reaction mixture followed by contacting the double-strandedamplification product with a second invasion-reaction mixture; saidfirst invasion-reaction mixture comprising a plurality of invasionprimers and no polymerase; and the second invasion-reaction mixturecomprises a plurality of deoxyribonucleotide triphosphate (dNTPs) and apolymerase.

Embodiment P52. The method of any one of Embodiment P1 to EmbodimentP51, wherein generating the first or second invasion strand comprisesthermally cycling between (i) about 67-80° C. for about 5 seconds toabout 30 seconds; and (ii) about 60-70° C. for about 30 to 90 seconds.

Embodiment P53. The method of any one of Embodiment P1 to EmbodimentP52, wherein the template polynucleotide comprises genomic DNA,complementary DNA (cDNA), cell-free DNA (cfDNA), messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free RNA (cfRNA), ornoncoding RNA (ncRNA).

Embodiment P54. The method of any one of Embodiment P1 to EmbodimentP52, wherein the template polynucleotide is about 100 to 1000nucleotides in length.

Embodiment P55. The method of any one of Embodiment P1 to EmbodimentP52, wherein the template polynucleotide is about 350 nucleotides inlength.

Embodiment P56. The method of any one of Embodiment P1 to EmbodimentP55, wherein the template polynucleotide and the double-strandedamplification product comprise known adapter sequences on the 5′ and 3′ends.

Embodiment P57. The method of any one of Embodiment P1 to EmbodimentP56, wherein generating a double-stranded amplification productcomprises bridge polymerase chain reaction (bPCR) amplification,solid-phase rolling circle amplification (RCA), solid-phase exponentialrolling circle amplification (eRCA), solid-phase recombinase polymeraseamplification (RPA), solid-phase helicase dependent amplification (HDA),template walking amplification, or emulsion PCR on particles, orcombinations of said methods.

Embodiment P58. The method of any one of Embodiment P1 to EmbodimentP56, wherein generating a double-stranded amplification productcomprises a bridge polymerase chain reaction (bPCR) amplification.

Embodiment P59. The method of any one of Embodiment P1 to EmbodimentP56, wherein generating a double-stranded amplification productcomprises a thermal bridge polymerase chain reaction (t-bPCR)amplification.

Embodiment P60. The method of any one of Embodiment P1 to EmbodimentP56, wherein generating a double-stranded amplification productcomprises a chemical bridge polymerase chain reaction (c-bPCR)amplification.

Embodiment P61. The method of any one of Embodiment P1 to EmbodimentP56, wherein generating a double-stranded amplification productcomprises amplifying the template polynucleotide or complement thereofon a solid support comprising a plurality of primers attached to saidsolid support, wherein the plurality of primers comprise a plurality offorward primers with complementarity to the template polynucleotide anda plurality of reverse primers with complementarity to a complement ofthe template polynucleotide, and the amplifying comprises a plurality ofcycles of strand denaturation, primer hybridization, and primerextension.

Embodiment P62. The method of Embodiment P61, wherein amplifyingcomprises incubation in a denaturant.

Embodiment P63. The method of Embodiment P62, wherein the denaturant isacetic acid, ethylene glycol, hydrochloric acid, nitric acid, formamide,guanidine, sodium salicylate, sodium hydroxide, dimethyl sulfoxide(DMSO), propylene glycol, urea, or a mixture thereof.

Embodiment P64. The method of any one of Embodiment P1 to EmbodimentP63, wherein the double-stranded amplification product is provided in aclustered array.

Embodiment P65. The method of any one of Embodiment P1 to EmbodimentP63, wherein the solid support is a bead.

Embodiment P66. The method of any one of Embodiment P1 to EmbodimentP63, wherein the solid support is substantially planar.

Embodiment P67. The method of any one of Embodiment P1 to EmbodimentP66, wherein the sequencing comprises sequencing by synthesis,sequencing by ligation, or pyrosequencing.

Embodiment P68. The method of any one of Embodiment P1 to EmbodimentP67, wherein generating a first sequencing read or a second sequencingread comprises a sequencing by synthesis process.

Embodiment P69. The method of any one of Embodiment P1 to EmbodimentP67, wherein generating a sequencing read comprises executing aplurality of sequencing cycles, each cycle comprising extending thesequencing primer by incorporating a nucleotide or nucleotide analogueusing a polymerase and detecting a characteristic signature indicatingthat the nucleotide or nucleotide analogue has been incorporated.

Embodiment P70. The method of Embodiment P69, further comprisingincorporating one or more unmodified dNTPs or one or more ddNTPs intothe 3′ end of the extended sequencing primer.

Embodiment P71. The method of Embodiment P6, wherein removing theinvasion strand comprises digesting the invasion strand using anexonuclease enzyme.

Embodiment P72. The method of Embodiment P6 or Embodiment P71, whereingenerating the second sequencing read comprises executing a plurality ofsequencing cycles, each cycle comprising extending the invasion primerby incorporating a nucleotide or nucleotide analogue using a polymeraseand detecting a characteristic signature indicating that the nucleotideor nucleotide analogue has been incorporated.

Embodiment P73. A substrate comprising: i) a first polynucleotideattached to the substrate; ii) a second polynucleotide attached to thesubstrate, wherein the second polynucleotide comprises a complementarysequence to the first polynucleotide; and iii) a third polynucleotidehybridized to the second polynucleotide, wherein the thirdpolynucleotide is not covalently attached to the substrate.

Embodiment P74. The substrate of Embodiment P73, wherein the thirdpolynucleotide comprises locked nucleic acids (LNAs), Bis-locked nucleicacids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridgednucleic acids (BNAs), 2′-O-methyl RNA:DNA chimeric nucleic acids, minorgroove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modifiedpyrimidine nucleic acids, peptide nucleic acids (PNAs), or combinationsthereof.

Embodiment P75. The substrate of Embodiment P73, wherein the thirdpolynucleotide comprises a homologous recombination complex comprising arecombinase bound thereto.

Embodiment P76. The substrate of Embodiment P75, wherein the homologousrecombination complex further comprises a loading factor, asingle-stranded binding (SSB) protein, or both.

Embodiment P77. The substrate of claim any one of Embodiment P73 toEmbodiment P76, further comprising a plurality of immobilizedoligonucleotides attached to the substrate via a linker.

Embodiment P78. The substrate of any one of Embodiment P73 to EmbodimentP77, wherein the substrate comprises a plurality of firstpolynucleotides attached to the substrate; a plurality of secondpolynucleotides attached to the substrate; and a plurality of thirdpolynucleotides hybridized to each of the second polynucleotides,wherein the plurality of third polynucleotides are not covalentlyattached to the substrate.

Embodiment P79. The substrate of any one of Embodiment P73 to EmbodimentP78, wherein the substrate comprises a glass surface comprising apolymer coating.

Embodiment P80. A method of reducing GC bias in a plurality ofsequencing reads, said method comprising sequencing a templatepolynucleotide to generate a plurality of sequencing reads according toEmbodiment P42.

Embodiment P81. A method of generating a template for a nucleic acidsequencing reaction, comprising: i) providing a solid support comprisinga plurality of immobilized oligonucleotide primers attached to the solidsupport via a linker, wherein the plurality of oligonucleotide primerscomprise a plurality of forward primers and a plurality of reverseprimers; ii) amplifying a template nucleic acid by using theoligonucleotide primers attached to the solid support to generate aplurality of double-stranded amplification products, eachdouble-stranded amplification product comprising a first strandhybridized to a second strand, wherein (a) each double-strandedamplification product comprises the template polynucleotide orcomplement thereof, and (b) the first strand and second strand are bothattached to the solid support; and iii) generating a first invasionstrand hybridized to the second strand by hybridizing one or moreinvasion primers to the second strand, and extending the one or moreinvasion primers, wherein the one or more invasion primers are notcovalently attached to the solid support; thereby generating a templatenucleic acid for a nucleic acid sequencing reaction.

Embodiment P82. The method of Embodiment P81, further comprisinghybridizing one or more sequencing primers to the first strand.

Embodiment P83. A method comprising: i) amplifying a template nucleicacid by using a plurality of oligonucleotide primers attached to a solidsupport to generate a plurality of double-stranded amplificationproducts, each double-stranded amplification product comprising a firststrand hybridized to a second strand, wherein the first strand andsecond strand are both attached to the solid support; and ii) generatinga first invasion strand hybridized to the second strand by hybridizingone or more invasion primers to the second strand, and extending the oneor more invasion primers to produce a single-stranded first strand,wherein the one or more invasion primers are not covalently attached tothe solid support.

ADDITIONAL EMBODIMENTS

The present disclosure provides the following additional illustrativeembodiments.

Embodiment 1. A method of sequencing a double-stranded polynucleotidecomprising a first strand hybridized to a second strand, wherein thefirst strand and second strand are both attached to a solid support,said method comprising: i) hybridizing an invasion primer to the secondstrand and extending the invasion primer with a polymerase, therebygenerating an invasion strand; ii) hybridizing a sequencing primer tothe first strand; iii) incorporating one or more nucleotides into thesequencing primer with a polymerase to create an extension strand; andiv) detecting the one or more incorporated nucleotides so as to identifyeach incorporated nucleotide in said extension strand, therebysequencing the first strand of the double-stranded polynucleotide.

Embodiment 2. The method of embodiment 1, further comprising removingthe first strand, removing the invasion strand, or both removing thefirst strand and removing the invasion strand.

Embodiment 3. The method of embodiment 1, further comprising removingthe invasion strand and hybridizing a second invasion primer to thefirst strand and extending the second invasion primer with a polymerase,thereby generating a second invasion strand.

Embodiment 4. A method of forming a plurality of single-strandedpolynucleotides attached to a solid support, said method comprising:contacting a plurality of double-stranded polynucleotides comprising afirst strand hybridized to a second strand with a plurality of invasionprimers, wherein the first strand and the second strand are attached tothe solid support; hybridizing one or more invasion primers to thesecond strand; and extending one or more invasion primers hybridized tothe second strand with a polymerase to generate one or more invasionstrands, displacing the first strand, thereby forming a plurality ofsingle-stranded polynucleotides attached to the solid support.

Embodiment 5. The method of embodiment 4, further comprising sequencingthe single-stranded polynucleotides.

Embodiment 6. The method of embodiment 4 or 5, further comprisingremoving the invasion strand and sequencing the second strand.

Embodiment 7. The method of any one of embodiments 1 to 6, comprisinghybridizing a second sequencing primer to the second strand andincorporating one or more nucleotides with a polymerase into the secondsequencing primer to create a second extension strand; and detecting theone or more incorporated nucleotides so as to identify each incorporatednucleotide in said second extension strand.

Embodiment 8. The method of embodiment 1, comprising nicking theinvasion strand to generate a 3′ end and incorporating one or morenucleotides into the 3′ end of the invasion primer with a polymerase tocreate an extension strand; and detecting the one or more incorporatednucleotides so as to identify each incorporated nucleotide in saidextension strand.

Embodiment 9. The method of any one of embodiments 2 to 8, whereinremoving the invasion strand comprises digesting the invasion strandusing an exonuclease enzyme.

Embodiment 10. The method of any one of embodiments 1 to 8, wherein thefirst strand is covalently attached to the solid support via a firstlinker and the second strand is covalently attached to the solid supportvia a second linker.

Embodiment 11. The method of any one of embodiments 1 to 10, wherein thedouble-stranded polynucleotides comprise known adapter sequences on the5′ and 3′ ends.

Embodiment 12. The method of any one of embodiments 1 to 11, wherein thesolid support comprises a plurality of polynucleotides, wherein eachpolynucleotide is attached to the solid support at a 5′ end of thepolynucleotide.

Embodiment 13. The method of any one of embodiments 1 to 12, wherein theinvasion primer comprises locked nucleic acids (LNAs), Bis-lockednucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs),bridged nucleic acids (BNAs), 2′-O-methyl RNA:DNA chimeric nucleicacids, minor groove binder (MGB) nucleic acids, morpholino nucleicacids, C5-modified pyrimidine nucleic acids, peptide nucleic acids(PNAs), phosphorothioate nucleic acids, or combinations thereof.

Embodiment 14. The method of any one of embodiments 1 to 12, wherein theinvasion primer comprises one or more locked nucleic acids (LNAs),2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalizedoligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs),peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC)nucleic acids.

Embodiment 15. The method of any one of embodiments 1 to 14, wherein theinvasion primer is about 15 to about 35 nucleotides in length.

Embodiment 16. The method of any one of embodiments 1 to 15, wherein theinvasion primer comprises one or more locked nucleic acids (LNAs) at the3′ end of the invasion primer sequence.

Embodiment 17. The method of any one of embodiments 1 to 16, furthercomprising contacting the invasion primer with a recombinase, a crowdingagent, a loading factor, a single-stranded binding (SSB) protein, or acombination thereof.

Embodiment 18. The method of any one of embodiments 1 to 17, whereingenerating the invasion strand comprises contacting the polynucleotidewith one or more invasion-reaction mixtures.

Embodiment 19. The method of embodiment 18, wherein each of theplurality of invasion-reaction mixtures comprise a plurality of invasionprimers, a plurality of deoxyribonucleotide triphosphate (dNTPs), apolymerase, or a combination thereof.

Embodiment 20. The method of embodiment 18 or 19, wherein each of theplurality of invasion-reaction mixtures comprise a denaturant,single-stranded DNA binding protein (SSB), or both a denaturant andsingle-stranded DNA binding protein (SSB).

Embodiment 21. The method of any one of embodiments 1 to 17, whereingenerating the invasion strand comprises a first plurality ofinvasion-primer extension cycles followed by a second plurality ofinvasion-primer extension cycles, wherein the reaction conditions forthe first plurality of invasion-primer extension cycles are differentthan the second plurality of invasion-primer extension cycles.

Embodiment 22. The method of embodiment 21, wherein the reactionconditions for the first plurality of invasion-primer extension cyclescomprise higher stringency hybridization conditions relative to thesecond plurality of invasion-primer extension cycles.

Embodiment 23. The method of any one of embodiments 1 to 17, whereingenerating the invasion strand comprises contacting the polynucleotidewith a buffered solution comprising dimethyl sulfoxide (DMSO), betaine,or a combination of dimethyl sulfoxide (DMSO) and betaine.

Embodiment 24. The method of any one of embodiments 1 to 23, whereinprior to hybridizing the invasion primer the method comprises amplifyingthe double-stranded polynucleotides with bridge polymerase chainreaction (bPCR) amplification, solid-phase rolling circle amplification(RCA), solid-phase exponential rolling circle amplification (eRCA),solid-phase recombinase polymerase amplification (RPA), solid-phasehelicase dependent amplification (HDA), template walking amplification,or emulsion PCR, or combinations of said methods.

Embodiment 25. The method of any one of embodiments 1 to 24, whereinsequencing comprises sequencing by synthesis, sequencing by binding,sequencing by ligation, or pyrosequencing.

Embodiment 26. The method of any one of embodiments 1 to 25, furthercomprising terminating extension by incorporating one or more unmodifieddNTPs or one or more ddNTPs into the 3′ end of the extension strand.

Embodiment 27. The method of any one of embodiments 1 to 26, comprisinghybridizing a second sequencing primer to the second strand andincorporating one or more nucleotides into the second sequencing primerwith a polymerase to create a second extension strand; and detecting theone or more incorporated nucleotides so as to identify each incorporatednucleotide in said second extension strand.

Embodiment 28. The method of any one of embodiments 1 to 25, comprising:terminating extension by incorporating one or more unmodified dNTPs orone or more ddNTPs into the 3′ end of the extension strand; hybridizinga second sequencing primer to the second strand and incorporating one ormore nucleotides into the second sequencing primer with a polymerase tocreate a second extension strand; and detecting the one or moreincorporated nucleotides so as to identify each incorporated nucleotidein said second extension strand; terminating extension by incorporatingone or more unmodified dNTPs or one or more ddNTPs into the 3′ end ofthe second extension strand; removing the invasion strand; hybridizing athird sequencing primer to the first strand and incorporating one ormore nucleotides into the third sequencing primer with a polymerase tocreate a third extension strand; and detecting the one or moreincorporated nucleotides so as to identify each incorporated nucleotidein said third extension strand; terminating extension by incorporatingone or more unmodified dNTPs or one or more ddNTPs into the 3′ end ofthe third extension strand; and hybridizing a fourth sequencing primerto the first strand and incorporating one or more nucleotides into thefourth sequencing primer with a polymerase to create a fourth extensionstrand; and detecting the one or more incorporated nucleotides so as toidentify each incorporated nucleotide in said fourth extension strand.

Embodiment 29. A substrate comprising: i) a first polynucleotideattached to the substrate; ii) a second polynucleotide attached to thesame substrate, wherein the second polynucleotide comprises acomplementary sequence to the first polynucleotide; and iii) a thirdpolynucleotide hybridized to the second polynucleotide, wherein thethird polynucleotide is not covalently attached to the substrate.

Embodiment 30. A method of sequencing a template polynucleotide, themethod comprising: (A) generating a double-stranded amplificationproduct comprising a first strand hybridized to a second strand, wherein(i) the double-stranded amplification product comprises the templatepolynucleotide or complement thereof, and (ii) the first strand andsecond strand are both attached to a solid support; (B) generating afirst invasion strand hybridized to the second strand by hybridizing aninvasion primer to the second strand, and extending the invasion primer,wherein the invasion primer is not covalently attached to the solidsupport; and (C) generating a first sequencing read by hybridizing oneor more sequencing primers to the first strand, and extending the one ormore first sequencing primers.

EXAMPLES Example 1. An Efficient Approach to Sequencing Two Strands ofthe Same Polynucleotide

Before a target nucleic acid is sequenced, some degree of DNApre-processing and converting it to a library molecule is typicallyrequired. For example, these steps may involve fragmenting inputpolynucleotides into an appropriate platform-specific size range,followed by an end-polishing step to generate blunt-ended DNA fragments.Common nucleic acid sequences (referred to as adapter sequences) on the3′ and 5′ ends are then ligated to these fragments. A functional librarymolecule typically includes the target molecule with specific adaptersequences added to the 3′ and 5′ ends, e.g., Illumina's P5 and P7adapters/primers, to ensure compatibility with the underlying flow cell,so it may be amplified appropriately. For example, typical platformprimers include 5′-AATGATACGGCGACCACCG (P5) (SEQ ID NO:34), or thecomplement thereof, and 5′-CAAGCAGAAGACGGCATACGA (P7) (SEQ ID NO:60), orthe complement thereof. An example of an adapter ligation protocolincludes phosphorylated template oligos at the 5′ end using a T4polynucleotide kinase in 1× T4 ligase buffer for 30 minutes at 37° C. ina thermocycler. The kinase is then denatured (e.g., by heating) and theoligo reaction mixture is slowly cooled to 20° C. (e.g., by slowlychanging the temperature by 0.1° C. every 2 seconds).

Current SBS platforms require clonal amplification of the initialtemplate library molecules to create clusters (i.e., polonies), eachcontaining 100s to 10,000s of forward and reverse copies of an initialtemplate library molecule, to increase the signal-to-noise ratio becausethe systems are not sensitive enough to detect the extension of one baseat the individual DNA template molecule level. Standard amplificationmethods employed in commercial sequencing devices (e.g., solid-phasebridge amplification) typically amplify a template using surfaceimmobilized primers to produce a plurality of double-stranded nucleicacid molecules, wherein at least one strand of each double-strandednucleic acid molecule is attached to the solid support at its 5′ ends. Acommon method of doing solid-phase amplification involves bridgeamplification methodologies (referred to as bridge PCR) as exemplifiedby the disclosures of U.S. Pat. Nos. 5,641,658; 7,115,400; 7,790,418;U.S. Patent Publ. No. 2008/0009420, each of which is incorporated hereinby reference in its entirety. In sum, bridge amplification methods allowamplification products (e.g., amplicons) to be immobilized on a solidsupport in order to form arrays comprised of colonies (or “clusters”) ofimmobilized nucleic acid molecules. Each cluster or colony on such anarray is formed from a plurality of identical immobilized polynucleotidestrands and a plurality of identical immobilized complementarypolynucleotide strands. The products of solid-phase amplificationreactions are referred to as “bridged” structures when formed byannealed pairs of immobilized polynucleotide strands and immobilizedcomplementary strands, both strands being immobilized on the solidsupport at the 5′ end, preferably via a covalent attachment. Duringbridge PCR, additional chemical additives may be included in thereaction mixture, in which the DNA strands are denatured by flowing adenaturant such as formamide or NaOH over the DNA, which chemicallydenatures complementary strands. This is followed by washing out thedenaturant and reintroducing a polymerase in buffer conditions thatallow primer annealing and extension.

Sequencing two strands of the sample dsDNA template, referred to aspaired-end, paired-strand, linked-strand, or dual-read sequencing, is apowerful technique to improve sequencing accuracy and is commonlyperformed in next-generation sequencing (NGS) workflows. Sequencing bysynthesis (SBS) is a common implementation of NGS and paired-endsequencing is typically performed on monoclonal clusters generated by aclonal amplification process. For example, nucleic acid libraries thathave common nucleic acid sequences (referred to as adapter sequences) onthe 3′ and 5′ ends of every library molecule are delivered into a flowcell. Within the flow cell are nucleic acid sequences (referred to asprimers) that are complementary to one or both of the adapter sequencesof the library molecules. The primers may be immobilized to a solidsupport (e.g., a flow cell or a bead); a solid support encompasses anytype of solid, porous, or hollow sphere, ball, cylinder, or othersimilar configuration composed of plastic, ceramic, metal, or polymericmaterial (e.g., hydrogel) onto which a nucleic acid may be immobilized(e.g., covalently or non-covalently). After hybridization of the adapterregion of a library nucleic acid molecule to the immobilizedoligonucleotides (i.e., primers) on the solid phase, a polymerase willmake an initial copy of the library nucleic acid molecule by extendingthe primer. The complement of the initial library molecule is nowattached to a solid support, and the initial library nucleic acidmolecules can either be removed from the flow cell, or can stay presentduring subsequent steps, depending on which clonal amplification methodis used. Next, spatially localized amplification of the initial singleseed molecule will occur by means of a solid-phase clonal amplificationprocess. Examples of clonal amplification techniques include, but arenot limited to, bridge PCR, solid-phase rolling circle amplification(RCA), solid-phase exponential rolling circle amplification, solid-phaserecombinase polymerase amplification (RPA), solid-phase helicasedependent amplification (HDA), template walking amplification, emulsionPCR on particles (beads), or combinations of the aforementioned methods.Optionally, during clonal amplification, additional solution-phaseprimers can be supplemented in the flow cell for enabling oraccelerating amplification.

It is typical for solid-phase clonal amplification to generatemonoclonal clusters that each consist of many double-stranded DNA(dsDNA) copies (10s to 100,000s) of the initially seeded library nucleicacid molecule. In SBS workflows, clusters of dsDNA are difficult tosequence effectively with high accuracy and read length, especially asminiaturization pushes the clusters to become more densely arranged on asolid support. To initiate an SBS sequencing reaction, a sequencingprimer needs to hybridize to a single-stranded region in the dsDNA andbe extended by a polymerase. Individual strands in dsDNA clusters aredifficult to access for hybridization of sequencing primers.Additionally, the polymerases used during SBS to incorporate 3′reversibly terminated nucleotides (dNTPs) or native dNTPs (for examplein pyrosequencing) typically do not have strand-displacementcapabilities, and so even if one is successful in incorporatingsequencing primers into dsDNA molecules, it is still challenging toextend said sequencing primers when the vast majority of DNA moleculesare in dsDNA format.

Due to these constraints, dsDNA amplicons in clusters are typicallyprocessed into single-stranded DNA (ssDNA), sometimes referred to aslinearization, by a variety of methods. The dsDNA structures may belinearized by cleavage of one or both strands with a restrictionendonuclease or by cleavage of one strand with a nicking endonuclease.Other methods of cleavage can be used as an alternative to restrictionenzymes or nicking enzymes, including chemical cleavage (e.g., cleavageof a diol linkage with periodate), cleavage of abasic sites by cleavagewith endonuclease, by exposure to heat or alkali, cleavage ofribonucleotides incorporated into amplification products otherwisecomprised of deoxyribonucleotides, photochemical cleavage, or cleavageof a peptide linker. Alternatively, the primers may be attached to thesolid support with a cleavable linker, such that upon exposure to acleaving agent, all or a portion of the primer is removed from thesurface. For example, one linearization method requires one or both ofthe immobilized primers to have a cleavable site, such as a uracil,diol, 8-oxoG, disulfide, photocleavable moieties, an RNA base or anendonuclease cleaving site. After the solid phase clonal amplificationprocess is complete, one of the two species of solid phase primers(either forward or reverse) can be cleaved (chemically, enzymatically oroptically), followed by a denaturation step to remove the cleavedmolecules. This transforms the dsDNA molecules into ssDNA moleculeswithin the cluster and provides a region available for hybridization ofa sequencing primer to initiate a sequencing reaction. The monoclonalclusters can proceed to any necessary post-processing steps such asblocking of free 3′ ends, removal of select amplicons, or hybridizationof a sequencing primer.

In conventional workflows, once ssDNA molecules are generated a firstsequencing read is performed by hybridizing a first sequencing primer toa complementary region (e.g., a region within the adapter portion) ofthe ssDNA molecule. In the presence of an enzyme (e.g., a DNApolymerase), nucleotides (e.g., labeled nucleotides) are incorporatedand detected such that the identity of the incorporated nucleotidesallows for the identification of the first strand. When the first readis complete (i.e., the first strand is read to a sufficient length withsufficient accuracy) the second strand that was initially cleaved duringlinearization must be regenerated prior to starting the second read.This can be done by additional amplification steps, such as additionalrounds of bridge PCR or another amplification process. Following anadditional amplification step after the first sequencing read, thesecond strand may then be sequenced. All of these steps add complexityand time to the DNA sequencing workflow and can also introduceadditional errors made by the polymerase used during solid phaseamplification. Highly accurate sequencing methods would greatly benefitfrom novel methods that bypass the need for additional amplificationsteps between the two sequencing reads of conventional paired-endsequencing workflows.

In accordance with various embodiments, the methods disclosed hereinpermit reading of the original first and second strands (e.g., the firstand second strand of the amplicons), reducing the time, reagents,expense, and risk of polymerase error inherent in previous methods.Importantly, methods described herein prevent the need for additionalsolid phase amplification between the two sequencing reads. Inembodiments, methods disclosed herein utilize strand invasion usinginvasion primers into dsDNA amplicons bound to a solid phase, followedby polymerase extension of the invasion primers. Strand invasion intodsDNA can be challenging in general, but can be particularly challengingin dense monoclonal clusters of dsDNA where DNA molecules are packedtightly together in a spatially localized fashion on a solid phase.Because the local concentration of full-length complementary strands isvery high, insertion of a traditional primer oligonucleotide isthermodynamically unfavorable.

The invasion primers are oligonucleotide sequences that binds to onestrand of the dsDNA molecule in the cluster. For example, the invasionprimer may bind to a portion of the common adapter sequence of only theforward, or only the reverse amplicons in clusters. These invasionoligonucleotides may include nucleic acids having a binding affinityhigher than the binding affinity of standard or canonical DNAoligonucleotides, such as locked nucleic acids (LNA), peptide nucleicacids (PNAs), 2′-O-methyl RNA:DNA chimeras, minor groove binder probes(MGB), or morpholino probes. The invasion primers are introduced into aflow cell that contains monoclonal dsDNA clusters generated using aknown amplification method or an amplification method described herein.Some of these invasion primers can undergo spontaneous strand invasioninto dsDNA, as is the case for example for PNA invasion primers underlow ionic strength conditions, while other invasion primers may needassistance of additives such as DMSO, ethylene glycol, formamide,betaine, or other denaturants that assist strand invasion by inducingmore breathability within dsDNA amplicons. For example, such additivesmay include a buffered solution containing about 0 to about 50% DMSO,about 0 to about 50% ethylene glycol, about 0 to about 20% formamide, orabout 0 to about 3M betaine. In order to achieve sufficient“breathability” within dsDNA amplicons that are bound to a solid phase,it is helpful to include additives that can assist the “fraying” of thedsDNA molecules, particularly at the 5′ and 3′ ends.

The invasion oligonucleotide can be introduced without a polymerase andallowed to invade and anneal to the complementary region, or it may beintroduced together with a polymerase for runoff extension. Examples ofpolymerases that can be used for runoff extension are strand-displacingpolymerases such as Bst large fragment, Bst2.0 (New England Biolabs),Bsm DNA polymerase, Bsu polymerase, SD polymerase, Vent exo-polymeraseor Phi29 polymerase. In certain experiments, it is preferable tointroduce the invasion oligonucleotide (e.g., a 15-75 bp invasionprimer) together with a polymerase in the same reaction mixture. Becauseof the close physical proximity of the forward and reverse strands ofthe dsDNA molecules within a cluster, the hybridization of the invasionoligo to one of the DNA strands is often transient, and can beoutcompeted easily by the reannealing of the full-length forward andreverse strands of the dsDNA molecules. To efficiently extend theinvasion oligos that transiently hybridize, it is useful to have thepolymerase within the same reaction mixture such that the polymerase canimmediately extend the invasion oligo during the transienthybridizations that occur. For example, we found that particularreaction conditions (30% DMSO and in presence of Bst LF polymerase anddNTPs) can enable efficient invasion and runoff extension of theinvasion oligo.

An example of the strand invasion and runoff method outlined above wasexecuted on dsDNA clusters that were generated with bridge PCR. ASalmonella genomic dsDNA library with an average library molecule sizeof approximately 350 bp was generated by using standard librarypreparation techniques. The dsDNA genomic library was introduced into aproprietary flow cell at a 1 μM concentration in presence of 5×SSCbuffer and 30% ethylene glycol. The flow cell was heated to 95° C. todenature the dsDNA library molecules, followed by cooling the flow cellto 45° C. to allow the denatured library molecules to bind to theimmobilized primers on the surface of the flow cell. A strand displacing(SD) polymerase (with SD polymerase buffer, 3 mM MgCl₂, and 0.2 mM ofeach dNTP) was subsequently introduced into the flow cell and heated to60° C. for 10 min to make an initial first copy of the library moleculesthat were hybridized to the flow cell primers. The initial librarymolecules were subsequently removed from the flow cell by flushing 0.1MNaOH through the lanes. This was followed by 45 bridge PCR cycles with aBst LF polymerase and formamide as a chemical denaturant, which werecyclically introduced into the flow cell. A positive control lane wassubsequently treated with USER enzyme mix which cleaved the forwardamplicons which contained a uracil base and were formed by extending theforward flow cell primers from the flow cell surface. A second lane ofthe flow cell did not go through cleavage protocols and instead wentthrough a strand invasion and runoff protocol, according to thefollowing steps.

For strand invasion and runoff, dsDNA clusters immobilized in a lane ofthe flow cell were exposed to a reagent mix that contained a pluralityof LNA invasion oligos (at 1 uM concentration) capable of invading andhybridizing to a portion of the common adapter sequence, 0.56 units/uLof Bst polymerase, 30% DMSO, and 0.2 mM of each dNTP. This reaction mixwas incubated at 65° C. for 5 min, followed by flowing in fresh reagentmix. A total of four distinct 5 min incubations with freshinvasion-reagent mixtures containing the LNA invasion oligonucleotides,Bst polymerase, DMSO and dNTPs was performed. Subsequently, we probedthe 3′ end of the ssDNA molecule of the cleaved positive control laneand the single-stranded fragment in the lanes that went through strandinvasion and runoff with a FAM-labeled DNA probe. For the strandinvasion and runoff conditions, we expect to see fluorescent signal ifstrand invasion and runoff is successful, since non-treated dsDNAclusters do not allow hybridization of the labeled DNA probe due toinaccessibility of the region to which the FAM-labeled probe wouldhybridize.

FIG. 2A shows fluorescence images of monoclonal clusters after FAM-probelabeling described above. Together, FIGS. 2A-2B show fluorescence imagesand data showing monoclonal DNA clusters examined under fourconditions: 1) non-cleaved, invaded/extended; 2) invasion oligo, noextension; 3) non-cleaved, no invasion/extension; 4) cleaved control.Condition 4 is a positive control whereby the clusters were convertedinto ssDNA by cleaving and removing one of the strands from the flowcell surface, followed by labeling the resulting ssDNA molecules with acomplementary FAM-labeled DNA probe. Condition 3 is a negative controlwhereby the amplicon clusters are not subjected to an invasion primernor extension conditions. Condition 2 reveals that an invasion primer iscapable of invading the double-stranded amplification product, butwithout extension of the invasion primer, the amplicons are notcompletely accessible to FAM-labeled probes. Condition 1 shows dsDNAclusters that were not cleaved, but were subjected to a method describedherein, such as strand invasion and extension, followed by hybridizationof a complementary FAM-labeled DNA probe to the liberated ssDNA strand.The probe is only able to hybridize if a complementary single-strandedregion is available. Following probe excitation and image acquisition,Condition 1 and condition 4 show the presence of punctate clustersindicative of successful ssDNA formation using the methods describedherein. The dsDNA clusters in condition 1 and condition 4 (positivecontrol) show clusters with similar fluorescence intensities and thenumber of identified features indicating successful probe binding tossDNA molecules in the monoclonal clusters; see FIG. 2B. For the dsDNAclusters subjected to the strand invasion conditions, the observedfluorescent signals are indicative of successful strand invasion andrunoff extension by a polymerase.

As described above, the amplification methods produced clusters ofoligonucleotides for sequencing. The initiation point for the firstsequencing reaction was provided by annealing a sequencing primercomplementary to a region within one of the strands. In the presence ofan enzyme (e.g., a DNA polymerase), nucleotides (e.g., labelednucleotides) are incorporated and detected such that the identity of theincorporated nucleotides allows for the identification of the firststrand. Thus, the first sequencing reaction may include hybridizing asequencing primer to a region of an amplification product, sequentiallyincorporating one or more nucleotides into a polynucleotide strandcomplementary to the region of amplified template strand to besequenced, identifying the base present in one or more of theincorporated nucleotide(s) and thereby determining the sequence of aregion of the template strand. Note, the second sequenced strand ispresent while sequencing the first strand, albeit the second strand ithybridized to the invasion strand.

The sequencing quality of the above-mentioned conditions were comparedto demonstrate the capability of the strand invasion and runoffconditions for generating sequenceable amplicons. To that end, theclusters of the cleaved control conditions and strand invasion/runoffconditions were compared in an SBS experiment. The appropriatesequencing primers were hybridized to the amplicons in lanes withcleaved control and strand invasion/runoff conditions, followed by aplurality of sequencing cycles on a proprietary sequencing instrument.Sequencing quality scores assign confidence to a particular base withina sequencing read by quantifying the probability that a base is calledincorrectly. As observed in FIG. 3, the quality scores remainedrelatively invariant for a plurality of sequencing cycles using theinvasion and sequencing methods as described herein. Quality scores wereslightly lower for sequencing on dsDNA clusters that went through strandinvasion and runoff as compared to sequencing on ssDNA amplicons thatwere generated by cleaving one of the two amplicons strands. Thecalculated accuracy for 55 cycles was 99.85% accuracy for the cleavedcontrol condition versus 99.80% for the strand invasion/runoffcondition.

Another example and subsequent experiment of the strand invasion andrunoff method outlined above was executed on dsDNA clusters that weregenerated with bridge PCR. In this experiment, following the firstsequencing read, the first strand is cleaved and removed prior tosequencing the second strand. A Salmonella genomic dsDNA library with anaverage library molecule size of approximately 350 bp was generated byusing standard library preparation techniques. The dsDNA genomic librarywas introduced into a proprietary flow cell at a 1 pM concentration inpresence of 5×SSC buffer and 30% ethylene glycol. The flow cell washeated to 95° C. to denature the dsDNA library molecules, followed bycooling the flow cell to 45° C. to allow the denatured library moleculesto bind to the immobilized primers on the surface of the flow cell. A SDpolymerase (with SD polymerase buffer, 3 mM MgCl₂, and 0.2 mM of eachdNTP) was subsequently introduced into the flow cell and heated to 60°C. for 10 min to make an initial first copy of the library moleculesthat were hybridized to the flow cell primers. The initial librarymolecules were subsequently removed from the flow cell by flushing 0.1 MNaOH through the lanes. This was followed by 45 bridge PCR cycles with aBst LF polymerase and formamide as a chemical denaturant, which werecyclically introduced into the flow cell. For strand invasion andrunoff, dsDNA clusters in a lane of the flow cell were exposed to areagent mix that contained a plurality of LNA invasion oligo (at 1 uMconcentration) capable of invading and hybridizing to a portion of thecommon adapter sequence, 0.56 units/uL of Bst polymerase, 30% DMSO, and0.2 mM of each dNTP. This reaction mix was incubated at 65° C. for 5min, followed by flowing in a reagent mixture containing LNA invasionoligo (at 1 uM concentration) capable of invading and hybridizing to aportion of the common adapter sequence, 0.56 units/uL of Bst polymerase,5% DMSO, 2.5M betaine, 25 ng/uL ET-SSB and 0.2 mM of each dNTP. Theselatter two steps were repeated once more.

The clusters underwent strand invasion and runoff steps, were hybridizedwith sequencing primers, and were sequenced for 100 bp to generate afirst sequencing read (i.e., a 100 bp sequencing read). This firstsequencing read was followed by USER-based cleavage of the strand (i.e.,the strand that was sequenced for the first sequencing read is cleaved),and denaturation of the strand together with the invasion/runoff productthat was hybridized to the reverse complement of the strand that wassequenced. Next, a second sequencing primer was hybridized to theremaining amplicons on the flow cell surface, followed by a 150 bpsecond sequencing read. The accuracy of the first sequencing read was99.756% with 99.3% of the reads mapped to the reference genome. Theaccuracy of the second sequencing read was 99.767% for the secondsequencing read while 99.7% of the reads mapped to the reference genome;see FIGS. 7A-7B. This example shows the capability of obtaininghigh-quality paired-end sequencing reads with the methodology describedherein.

Example 2: PNAs within Invasion Primers

Peptide nucleic acids (PNAs) can be used as invasion oligonucleotides inanother example of this invention; see the schematic illustrated inFIGS. 4A-4B. Peptide nucleic acids consist of a pseudopeptide backbone,which has been shown to be capable of invading dsDNA. MiniPEG-γPNAs areparticularly beneficial because they have better water solubility (Bahalet al. ChemBioChem, 13(1), 56-60). PNAs typically do not have a 3′-OHthat is extendible by a DNA polymerase, though one can consider PNA-DNAchimeras that have 3-7 bp of canonical DNA nucleotides at the 3′ end ofthe oligonucleotide to be extendable by a DNA polymerase. MiniPEG-γPNAscan be designed to invade into dsDNA clusters by targeting a sequenceregion in the common adapter sequence of all clusters. PNAs can bedesigned for strand invasion into any part of the common adaptersequences, but targeting near the 5′ end of one of the amplicons isbeneficial because it renders the complementary strand available forhybridization of another oligonucleotide, as shown in FIG. 4A. A secondinvasion oligonucleotide that hybridizes on the “liberated” ssDNAfragment opposite of the PNA invasion site can then be extended by astrand-displacing DNA polymerase. As a result, one of the two strands ofevery dsDNA duplex has now been rendered into a ssDNA fragment that canbe sequenced by hybridizing a sequencing primer followed a plurality ofsequencing reactions.

Example 3: Recombinase-Assisted Invasion

Another possibility for enabling strand invasion into dsDNA molecules inmonoclonal clusters is by using a recombinase enzyme that enables theinsertion of a DNA oligonucleotide complementary to part of the commonadapter sequence; see FIGS. 5A-5B. A reagent mixture consisting of aninvasion oligonucleotide, a recombinase, and necessary cofactors forforming a pre-synaptic filament (i.e. an oligonucleotide complexed withrecombinase enzymes) is flowed into the flow cell that contains dsDNAclusters. The pre-synaptic filaments search the dsDNA molecules inmonoclonal clusters until homology is found, after which the invasionoligonucleotide is inserted into the dsDNA to form a D-loop. Afterstrand invasion, a strand-displacing polymerase can be introduced thatextends the invasion oligonucleotide, thereby rendering the oppositestrand of the original dsDNA duplex into a single-stranded form. ThessDNA molecule that is generated is then available for hybridization ofa sequencing primer and the subsequent start of a first sequencing read.Examples of recombinases include, but are not limited to, T4 UvsX (andpossibly its cofactor UvsY, and single-stranded binding protein gp32),Rad51, and RecA. The recombinase can be present in the same reaction mixas the strand-displacing polymerase, or the strand-displacing polymerasecan be introduced after strand invasion with the recombinase has beendone first.

Example 4. Limiting GC Bias in Sequencing Reads

When generating sequencing reads it is advantageous to obtain as broad arepresentation of the genome as possible. It is known that PCRamplification can introduce artifacts into sequencing libraries. Inaddition to nucleotide misincorporation, PCR amplification tends to beuneven, so that some sequence species become overrepresented in theresulting library. This situation is exacerbated by templates withGC-biased compositions. It is well known that extreme base compositions,i.e., GC-poor or GC-rich sequences, lead to uneven coverage or even nocoverage of reads across the entirety of genome (see, for example Quailet al. Nat Methods. 2008 December; 5(12):1005-10). For example, readcoverage of sequenced regions may be biased depending on the GC contentof the library, when it was found the highest read density was found inintervals with elevated GC content (Dohm et al. Nucleic Acids Res. 2008September; 36(16):e105). This GC bias can be introduced during PCRamplification of the library, cluster amplification, and the sequencingreactions. New experimental designs and optimized amplificationprotocols have been developed to reduce GC bias, and so it is preferablethat sequencing reactions do not introduce any GC bias.

To minimize any GC bias, we discovered that it is beneficial to use atleast two distinct invasion-reaction mixtures in order to obtain arecipe that works well for invasion/runoff for a wide range of GC %within the inserts of the sequencing library. For example,invasion/runoff with 30% DMSO may generate runoff extension productsthat are stable enough to stay hybridized in presence of 30% DMSO,whereas invasion/runoff products for inserts that have <30% GC contentmay not be stable enough to stay hybridized in that particular reactionmixture. In order to mitigate this and enable successful invasion/runoffacross a wider range of GC % for the inserts of the sequencing library,one can exchange the solution of 30% DMSO, invasion oligonucleotide andpolymerase for a solution that contains 5% DMSO, invasionoligonucleotide and polymerase, for example. In some experiments,cycling through different invasion-reaction mixtures (e.g., a firstinvasion-reaction mixture, followed by a second invasion-reactionmixture, followed by the first invasion-reaction mixture, etc.) limitedthe GC bias.

An example demonstrating the importance of modulating the invasion andrunoff condition for obtaining even GC coverage representation in DNAsequencing is shown in FIG. 6. The same experimental conditions asoutlined in the examples above were used in two lanes of the flow cell:(i) a positive control lane (lane #1) was treated with USER enzyme mixafter cluster amplification, which cleaved the forward amplicons (whichcontained a uracil base and were formed by extending the forward flowcell primers) from the flow cell surface, and (ii) another lane (lane#2) of the flow did not go through cleavage of any of the flow cellprimers and instead went through a strand invasion and runoff protocol,based on strand invasion and runoff in presence of 30% DMSO, asmentioned above. In a third lane (lane #3) of the flow cell, theinvasion and runoff reaction was split up into two different steps: (i)one step was identical to the invasion and runoff conditions in lane #2,and this was followed by (ii) an additional step of invasion and runoffconditions with 5% DMSO and 2.5M betaine. The resulting data in FIG. 6shows the benefit of performing a plurality of distinct invasionreaction cycles, resulting in a much broader coverage of the range of GCcontent. The invasion and runoff reactions performed first in 30% DMSO,and followed by an additional invasion and runoff step in 5% DMSO and2.5M betaine (lane #3), showed a similar GC coverage bias profilecompared to the positive control lane that received cleavage of theforward amplicons (lane #1), and had much more even coverage compared tothe condition that used 30% DMSO only (lane #2). The Salmonella genomiclibrary, which was used for this experiment, has a genome with a GCcontent ranging from approximately 15% to approximately 85%, and the GCcoverage is targeted to be as uniform as possible (normalized to 1)across this range. The spikes that are observed at the low and high endsof the GC-range are due to the relatively low coverage (low sampling) inthis particular experiment and the low representation of the extreme GCcontent fragments (<20% and >75%) in this genome. Having greatercoverage of reads across the entirety of the genome aids mapping and SNPcalling, and makes assembly more straightforward.

Example 5. Additional Approaches to Sequencing Two Strands of the SamePolynucleotide

Sequencing two strands of the sample dsDNA template is a powerfultechnique to improve sequencing accuracy and is commonly performed innext-generation sequencing (NGS) workflows. Sequencing by synthesis(SBS) is a common implementation of NGS and paired-end sequencing istypically performed on monoclonal clusters generated by a clonalamplification process. It is typical for solid-phase clonalamplification to generate monoclonal clusters that each consist of manydouble-stranded DNA (dsDNA) copies (10s to 100,000s) of the initiallyseeded library nucleic acid molecule. In SBS workflows, clusters ofdsDNA are difficult to sequence effectively with high accuracy and readlength, especially as miniaturization pushes the clusters to become moredensely arranged on a solid support. To initiate an SBS sequencingreaction, a sequencing primer needs to hybridize to a single-strandedregion in the dsDNA and be extended by a polymerase. Individual strandsin dsDNA clusters are difficult to access for hybridization ofsequencing primers. Additionally, the polymerases used during SBS toincorporate 3′ reversibly terminated nucleotides (dNTPs) or native dNTPs(for example in pyrosequencing) typically do not havestrand-displacement capabilities, and so even if one is successful inincorporating sequencing primers into dsDNA molecules, it is stillchallenging to extend said sequencing primers when the vast majority ofDNA molecules are in dsDNA format.

Due to these constraints, dsDNA amplicons in clusters are typicallyprocessed into single-stranded DNA (ssDNA), sometimes referred to aslinearization, by a variety of methods. The dsDNA structures may belinearized by cleavage of one or both strands with a restrictionendonuclease or by cleavage of one strand with a nicking endonuclease.Other methods of cleavage can be used as an alternative to restrictionenzymes or nicking enzymes, including chemical cleavage (e.g., cleavageof a diol linkage with periodate), cleavage of abasic sites by cleavagewith endonuclease, by exposure to heat or alkali, cleavage ofribonucleotides incorporated into amplification products otherwisecomprised of deoxyribonucleotides, photochemical cleavage, or cleavageof a peptide linker. Alternatively, the primers may be attached to thesolid support with a cleavable linker, such that upon exposure to acleaving agent, all or a portion of the primer is removed from thesurface. For example, one linearization method requires one or both ofthe immobilized primers to have a cleavable site, such as a uracil,diol, 8-oxoG, disulfide, photocleavable moieties, an RNA base or anendonuclease cleaving site. After the solid phase clonal amplificationprocess is complete, one of the two species of solid phase primers(either forward or reverse) can be cleaved (chemically, enzymatically oroptically), followed by a denaturation step to remove the cleavedmolecules. This transforms the dsDNA molecules into ssDNA moleculeswithin the cluster and provides a region available for hybridization ofa sequencing primer to initiate a sequencing reaction. The monoclonalclusters can proceed to any necessary post-processing steps such asblocking of free 3′-OH ends, removal of select amplicons, orhybridization of a sequencing primer.

In conventional workflows, once ssDNA molecules are generated a firstsequencing read is performed by hybridizing a first sequencing primer toa complementary region (e.g., a region within the adapter portion) ofthe ssDNA molecule. In the presence of an enzyme (e.g., a DNApolymerase), nucleotides (e.g., labeled nucleotides) are incorporatedand detected such that the identity of the incorporated nucleotidesallows for the identification of the first strand. When the first readis complete (i.e., the first strand is read to a sufficient length withsufficient accuracy) the second strand that was initially cleaved duringlinearization must be regenerated prior to starting the second read.This can be done by additional amplification steps, such as additionalrounds of bridge PCR or another amplification process. Following anadditional amplification step after the first sequencing read, thesecond strand may then be sequenced. All of these steps add complexityand time to the DNA sequencing workflow and can also introduceadditional errors made by the polymerase used during solid phaseamplification. Highly accurate sequencing methods would greatly benefitfrom novel methods that bypass the need for additional amplificationsteps between the two sequencing reads of conventional paired-endsequencing workflows.

In accordance with various embodiments, the methods disclosed hereinpermit reading of the original first and second strands (e.g., the firstand second strand of the amplicons), reducing the time, reagents,expense, and risk of polymerase error inherent in previous methods.Importantly, methods described herein prevent the need for additionalsolid phase amplification between the two sequencing reads. Inembodiments, methods disclosed herein utilize strand invasion usinginvasion primers into dsDNA amplicons bound to a solid phase, followedby polymerase extension of the invasion primers. Strand invasion intodsDNA can be challenging in general, but can be particularly challengingin dense monoclonal clusters of dsDNA where DNA molecules are packedtightly together in a spatially localized fashion on a solid phase.Because the local concentration of full-length complementary strands isvery high, insertion of a traditional primer oligonucleotide isthermodynamically unfavorable.

The invasion primers are oligonucleotide sequences that binds to onestrand of the dsDNA molecule in the cluster. For example, the invasionprimer may bind to a portion of the common adapter sequence of only theforward, or only the reverse amplicons in clusters. These invasionoligonucleotides may include nucleic acids having a binding affinityhigher than the binding affinity of standard or canonical DNAoligonucleotides, such as locked nucleic acids (LNA), peptide nucleicacids (PNAs), 2′-O-methyl RNA:DNA chimeras, minor groove binder probes(MGB), or morpholino probes. The invasion primers may include one ormore deoxyuracils (dUs). The invasion primers may include one or morephosphorothioate groups. The invasion primers are introduced into a flowcell that contains monoclonal dsDNA clusters generated using a knownamplification method or an amplification method described herein. Someof these invasion primers can undergo spontaneous strand invasion intodsDNA, as is the case for example for PNA invasion primers under lowionic strength conditions, while other invasion primers may needassistance of additives such as DMSO, ethylene glycol, formamide,betaine, or other denaturants or additives that assist strand invasionby inducing more breathability within dsDNA amplicons. For example, suchadditives may include a buffered solution containing about 0 to about50% DMSO, about 0 to about 50% ethylene glycol, about 0 to about 20%formamide, or about 0 to about 3M betaine. In order to achievesufficient “breathability” within dsDNA amplicons that are bound to asolid phase, it is helpful to include additives that can assist the“fraying” of the dsDNA molecules, particularly at the 5′ and 3′ ends.

As described herein and in Example 1, the invasion oligonucleotide canbe introduced without a polymerase and allowed to invade and anneal tothe complementary region, or it may be introduced together with apolymerase for runoff extension. In certain experiments, it ispreferable to introduce the invasion oligonucleotide (e.g., a 15-75 bpinvasion primer) together with a polymerase in the same reactionmixture. Because of the close physical proximity of the forward andreverse strands of the dsDNA molecules within a cluster, thehybridization of the invasion oligo to one of the DNA strands is oftentransient, and can be outcompeted easily by the reannealing of thefull-length forward and reverse strands of the dsDNA molecules. Toefficiently extend the invasion oligos that transiently hybridize, it isuseful to have the polymerase within the same reaction mixture such thatthe polymerase can immediately extend the invasion oligo during thetransient hybridizations that occur.

The initiation point for the first sequencing reaction is provided byannealing a sequencing primer complementary to a region within one ofthe strands. In the presence of an enzyme (e.g., a DNA polymerase),nucleotides (e.g., labeled nucleotides) are incorporated and detectedsuch that the identity of the incorporated nucleotides allows for theidentification of the first strand. Thus, the first sequencing reactionmay include hybridizing a sequencing primer to a region of anamplification product, sequentially incorporating one or morenucleotides into a polynucleotide strand complementary to the region ofamplified template strand to be sequenced, identifying the base presentin one or more of the incorporated nucleotide(s) and thereby determiningthe sequence of a region of the template strand. Note, the secondsequenced strand is present while sequencing the first strand, albeitthe second strand is hybridized to the invasion strand.

Additional embodiments of methods of paired-strand sequencing by strandinvasion of an invasion primer are disclosed herein. FIG. 8A illustratesan invasion primer annealed to the 3′ end of one of the strands. Inembodiments, the invasion primer includes one or more 5′phosphorothioate groups (e.g., 3-5 phosphorothioate linking groups) toprotect from exonuclease digestion. In embodiments, the invasion primerfurther includes a cleavable site (e.g., a 3′ deoxyuracil triphosphate(dUTP)). After runoff extension of the invasion oligonucleotide has beencompleted, one strand of the initial dsDNA molecule is nowsingle-stranded and available for a first sequencing read, as shown inFIG. 8B. This renders one of the two strands of the original dsDNAamplicon available for hybridization of a sequencing primer to initiatethe SBS process. The sequenced strand may optionally further be cleavedat a cleavable site (represented as ‘X’) and removed, thus leaving thecomplementary strand available for sequencing, as illustrated in FIG.8B. Subsequently, the 3′ end of the invasion primer may be cleaved at acleavable site (e.g., nicked at the dU), leaving behind a 5′-phosphatein the extended part of the invasion strand that can subsequently bedegraded with a 5′ to 3′ exonuclease, allowing for the invasion primerto serve as a sequencing primer for the second strand, as illustrated inFIGS. 8C-8D. In some embodiments, the invasion primer is treated with a3′ phosphatase (for example Endonuclease IV or PNK) to generate a 3′hydroxyl group prior to sequencing.

In a modified embodiment of the above method, once runoff extension ofthe invasion oligonucleotide has been completed, one strand of theinitial dsDNA molecule is now single-stranded and available for a firstsequencing read, as shown in FIG. 9B. This renders one of the twostrands of the original dsDNA amplicon available for hybridization of asequencing primer to initiate the SBS process. The sequenced strand mayfurther be extended with natural dNTPs after sequencing the first readto complete the extension of the sequenced strand, as illustrated inFIG. 9B, thereby preventing any rehybridization of any non-sequencedamplicon to the complement. Subsequently, the 3′ end of the invasionprimer may be cleaved at a cleavable site (e.g., nicked at the dU andremoved), leaving behind a 5′-phosphate in the invasion strand that cansubsequently be degraded with a 5′ to 3′ exonuclease, allowing for theinvasion primer to serve as a sequencing primer for the second strand,as illustrated in FIGS. 9C-9D. Alternatively, the sequenced strand mayfurther be extended with a one or more ddNTPs to prevent furtherextension, as illustrated in FIG. 10B. Subsequently, the 3′ end of theinvasion primer may be cleaved at a cleavable site (e.g., nicked at thedU and removed), leaving behind a 5′-phosphate in the invasion strandthat can subsequently be degraded with a 5′ to 3′ exonuclease, allowingfor the invasion primer to serve as a sequencing primer for the secondstrand, as illustrated in FIGS. 10C-10D. In some embodiments, theinvasion primer is treated with a 3′ phosphatase (for exampleEndonuclease IV or PNK) to generate a 3′ hydroxyl group prior tosequencing. Advantageously, neither of these two embodiments require theremoval of the first sequenced strand, further reducing cost and timerequired for high-accuracy paired-strand sequencing.

As an alternative to digesting away the invasion strand prior tosequencing the second strand, internal cleavable sites (e.g., cleavableinternucleosidic bonds) may be introduced into the invasion strand. Asin the methods shown supra, FIG. 11A illustrates an embodiment whereinthe invasion primer is annealed to the 3′ end of one of the strands. Inthe embodiment depicted in FIG. 11A, the invasion primer includes one ormore phosphorothioate nucleic acids at the 5′ end to protect fromexonuclease digestion, and a cleavable site at the 3′ end (e.g., one ormore deoxyuracil nucleobases). Runoff extension of the invasionoligonucleotide is then performed with an amplification mixture thatprovides cleavable sites (e.g., a mixture of dUTP, dATP, dGTP, and dCTPnucleotides) leaving one strand of the initial dsDNA moleculesingle-stranded and available for a first sequencing read, as shown inFIG. 11B. The sequenced strand may optionally further be cleaved at acleavable site (represented as ‘X’) and removed, thus leaving thecomplementary strand available for sequencing, as illustrated in FIG.11B. Subsequently, the invasion strand may be cleaved at internalcleavable sites (e.g., cleaved at the dU sites), leaving behind small,low Tm fragments (e.g., melting temperatures in the range of 0° C. toabout 60° C.) that may be thermally denatured away, as shown in FIG.11C. Additionally, this cleavage and denaturation step exposes the 3′end of the invasion oligo, allowing for the invasion primer to serve asa sequencing primer for the second strand, as illustrated in FIGS.11C-11D. In some embodiments, the invasion primer is treated with a 3′phosphatase (for example Endonuclease IV or PNK) to generate a 3′hydroxyl group prior to sequencing.

As an additional alternative to digesting away the invasion strand priorto sequencing the second strand, internal cleavable sites (e.g.,cleavable internucleosidic bonds) may be introduced into the invasionstrand. Following cleavage, the small products annealed to the secondstrand may then be digested away. As in the methods shown supra, FIG.12A illustrates an invasion primer annealed to the 3′ end of one of thestrands. In embodiments, the invasion primer includes one or morephosphorothioate group(s) towards the 5′ end to protect the invasionprimer from 5′ to 3′ exonuclease digestion. In embodiments, the invasionprimer also includes a cleavable site (also referred to herein as ascissile linkage). For example, as depicted as a ‘U’, the cleavable sitemay be a deoxyuracil (dU) towards the 3′ end of the invasion oligo.Runoff extension of the invasion oligonucleotide is then performed withdUTP, dATP, dGTP, and dCTP, leaving one strand of the initial dsDNAmolecule single-stranded and available for a first sequencing read, asshown in FIG. 12B. Once the first sequencing read has been obtained, the3′ end of the first sequencing read is capped by ddNTP incorporation. Asecond sequencing read is then obtained by annealing and extending asecond sequencing primer 3′ of the terminated first sequencing read.Subsequently, a ddNTP is incorporated into the 3′ end of the secondsequencing read, and thereafter the invasion strand may be nicked atinternal scissile sites (e.g., resulting from amplification with thedUTP), leaving behind small fragments with exposed 5′ ends that may beremoved under suitable conditions, for example, by lambda exonucleasedigestion, as shown in FIGS. 12C-12D. This cleavage and removal stepexposes the 3′ end of the second strand, making it available for a thirdsequencing read, as shown in FIG. 12E. Once the third sequencing readhas been obtained, the 3′ end of the third sequencing read is capped byddNTP incorporation. A fourth sequencing read is then obtained byannealing and extending a fourth sequencing primer 3′ of the terminatedthird sequencing read, as illustrated in FIG. 12E.

What is claimed is:
 1. A method of sequencing a polynucleotidecomprising a first strand hybridized to a second strand, wherein thefirst strand and second strand are both attached to a solid support,said method comprising: i) hybridizing an invasion primer to the secondstrand and extending the invasion primer with a polymerase, therebygenerating an invasion strand and a single-stranded first strand,wherein the invasion primer is not covalently attached to the solidsupport; ii) hybridizing a sequencing primer to the single-strandedfirst strand; iii) incorporating one or more nucleotides into thesequencing primer with a polymerase to create an extension strand; andiv) detecting the one or more incorporated nucleotides so as to identifyeach incorporated nucleotide in said extension strand, therebysequencing the first strand of the polynucleotide.
 2. The method ofclaim 1, further comprising removing the first strand, removing theinvasion strand, or both removing the first strand and removing theinvasion strand.
 3. The method of claim 1, further comprising removingthe invasion strand and hybridizing a second invasion primer to thefirst strand and extending the second invasion primer with a polymerase,thereby generating a second invasion strand and a single-stranded secondstrand, wherein the second invasion primer is not covalently attached tothe solid support.
 4. The method of claim 1, further comprising nickingthe invasion strand at a cleavable site to generate a 3′ end andincorporating one or more nucleotides into the 3′ end of the nickedinvasion strand with a polymerase to create a second extension strand;and detecting the one or more incorporated nucleotides so as to identifyeach incorporated nucleotide in said second extension strand.
 5. Themethod of claim 2, wherein removing the invasion strand comprisesdigesting the invasion strand using an exonuclease enzyme.
 6. The methodof claim 1, wherein the first strand is covalently attached to the solidsupport via a first linker and the second strand is covalently attachedto the solid support via a second linker.
 7. The method of claim 1,wherein the polynucleotide comprise known adapter sequences on the 5′and 3′ ends.
 8. The method of claim 1, wherein the solid supportcomprises a plurality of polynucleotides, wherein each polynucleotide isattached to the solid support at a 5′ end of the polynucleotide.
 9. Themethod of claim 1, wherein the invasion primer comprises locked nucleicacids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalatingnucleic acids (TINAs), bridged nucleic acids (BNAs), 2′-O-methyl RNA:DNAchimeric nucleic acids, minor groove binder (MGB) nucleic acids,morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptidenucleic acids (PNAs), phosphorothioate nucleic acids, or combinationsthereof.
 10. The method of claim 1, wherein the invasion primercomprises one or more locked nucleic acids (LNAs),2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalizedoligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs),peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC)nucleic acids.
 11. The method of claim 1, wherein the invasion primer isabout 15 to about 35 nucleotides in length.
 12. The method of claim 1,wherein the invasion primer comprises one or more locked nucleic acids(LNAs) at the 3′ end of the invasion primer sequence.
 13. The method ofclaim 1, further comprising contacting the invasion primer with arecombinase, a crowding agent, a loading factor, a single-strandedbinding (SSB) protein, or a combination thereof.
 14. The method of claim1, wherein generating the invasion strand comprises contacting thepolynucleotide with one or more invasion-reaction mixtures.
 15. Themethod of claim 14, wherein each of the plurality of invasion-reactionmixtures comprise a plurality of invasion primers, a plurality ofdeoxyribonucleotide triphosphate (dNTPs), a polymerase, or a combinationthereof.
 16. The method of claim 14, wherein each of the plurality ofinvasion-reaction mixtures comprise a denaturant, single-stranded DNAbinding protein (SSB), or both a denaturant and single-stranded DNAbinding protein (SSB).
 17. The method of claim 1, wherein generating theinvasion strand comprises a first plurality of invasion-primer extensioncycles followed by a second plurality of invasion-primer extensioncycles, wherein the reaction conditions for the first plurality ofinvasion-primer extension cycles are different than the second pluralityof invasion-primer extension cycles.
 18. The method of claim 17, whereinthe first plurality of invasion-primer extension cycles comprisesincubation in a first denaturant and the second plurality ofinvasion-primer extension cycles comprises incubation in a seconddenaturant, wherein each of the first and second denaturant comprisesadditives, and wherein the concentrations of the additives in the firstdenaturant is higher than the concentrations of the additives in thesecond denaturant.
 19. The method of claim 1, wherein generating theinvasion strand comprises contacting the polynucleotide with a bufferedsolution comprising dimethyl sulfoxide (DMSO), betaine, or a combinationof dimethyl sulfoxide (DMSO) and betaine.
 20. The method of claim 1,wherein prior to hybridizing the invasion primer the method comprisesamplifying the polynucleotide with bridge polymerase chain reaction(bPCR) amplification, solid-phase rolling circle amplification (RCA),solid-phase exponential rolling circle amplification (eRCA), solid-phaserecombinase polymerase amplification (RPA), solid-phase helicasedependent amplification (HDA), template walking amplification, oremulsion PCR, or combinations of said methods.
 21. The method of claim1, wherein sequencing comprises sequencing by synthesis, sequencing bybinding, sequencing by ligation, or pyrosequencing.
 22. The method ofclaim 1, further comprising terminating extension by incorporating oneor more unmodified dNTPs or one or more ddNTPs into the 3′ end of theextension strand.
 23. The method of claim 1, further comprising removingsaid invasion strand; hybridizing a second sequencing primer to thesecond strand and incorporating one or more nucleotides into the secondsequencing primer with a polymerase to create a second extension strand;and detecting the one or more incorporated nucleotides so as to identifyeach incorporated nucleotide in said second extension strand.
 24. Themethod of claim 1, comprising: terminating extension by incorporatingone or more unmodified dNTPs or one or more ddNTPs into the 3′ end ofthe extension strand; hybridizing a second sequencing primer to thesecond strand and incorporating one or more nucleotides into the secondsequencing primer with a polymerase to create a second extension strand;and detecting the one or more incorporated nucleotides so as to identifyeach incorporated nucleotide in said second extension strand;terminating extension by incorporating one or more unmodified dNTPs orone or more ddNTPs into the 3′ end of the second extension strand;removing the invasion strand; hybridizing a third sequencing primer tothe first strand and incorporating one or more nucleotides into thethird sequencing primer with a polymerase to create a third extensionstrand; and detecting the one or more incorporated nucleotides so as toidentify each incorporated nucleotide in said third extension strand;terminating extension by incorporating one or more unmodified dNTPs orone or more ddNTPs into the 3′ end of the third extension strand; andhybridizing a fourth sequencing primer to the first strand andincorporating one or more nucleotides into the fourth sequencing primerwith a polymerase to create a fourth extension strand; and detecting theone or more incorporated nucleotides so as to identify each incorporatednucleotide in said fourth extension strand.
 25. The method of claim 3,further comprising hybridizing a second sequencing primer to thesingle-stranded second strand and incorporating one or more nucleotidesinto the second sequencing primer with a polymerase to create a secondextension strand; and detecting the one or more incorporated nucleotidesso as to identify each incorporated nucleotide in said second extensionstrand.
 26. The method of claim 1, further comprising removing the firststrand by cleaving the first strand at a cleavable site, washing awaythe cleaved strand, and generating a second sequencing read byhybridizing one or more second sequencing primers to the second strand;and extending the one or more second sequencing primers.
 27. The methodof claim 1, wherein the solid support comprises a plurality ofimmobilized primers.
 28. The method of claim 27, wherein prior togenerating the first invasion strand, the method comprises removingimmobilized primers that do not contain a first or a second strand. 29.The method of claim 1, wherein extending the invasion primer comprisesincorporating one or more deoxyuracil nucleobases.
 30. A method ofsequencing a polynucleotide comprising a first strand hybridized to asecond strand, wherein the first strand and second strand are bothattached to a solid support, said method comprising: i) hybridizing afirst invasion primer to the second strand and extending the firstinvasion primer with a polymerase, thereby generating a first invasionstrand and a single-stranded first strand, wherein the first invasionprimer is not covalently attached to the solid support; ii) hybridizinga first sequencing primer to the single-stranded first strand; iii)incorporating one or more nucleotides into the sequencing primer with apolymerase to create a first extension strand; iv) detecting the one ormore incorporated nucleotides so as to identify each incorporatednucleotide in said first extension strand, thereby sequencing the firststrand of the polynucleotide; v) removing the first invasion strand andhybridizing a second invasion primer to the first strand and extendingthe second invasion primer with a polymerase, thereby generating asecond invasion strand and a single-stranded second strand, wherein thesecond invasion primer is not covalently attached to the solid support;vi) hybridizing a second sequencing primer to the single-stranded secondstrand; vii) incorporating one or more nucleotides into the secondsequencing primer with a polymerase to create a second extension strand;and viii) detecting the one or more incorporated nucleotides so as toidentify each incorporated nucleotide in said second extension strand,thereby sequencing the second strand of the polynucleotide.